Simultaneous determination of cyromazine,melamine and their biodegradation products by ion-pair high-performance liquid chromatography

An ion-pair high-performance liquid chromatography with ultraviolet detection method for the determination of cyromazine, melamine and its biodegradation products (ammeline, ammelide, cyanuric acid and biuret) was developed. C18 column was utilised to separate the six analytes with a mobile phase consisting of perchloric acid-ammonia solution and acetonitrile, under gradient elution and variable flow rate. The detection wavelengths were 205 nm for cyanuric acid and biuret and 222 nm for cyromazine, melamine, ammeline and ammelide. For analysis of sediment samples, the extraction solution containing acetonitrile, ammonia and water (80:10:10 by volume) was used to extract the analytes from sediment matrix. Using the extraction method for the spiked sediment sample, high linearity of matrix-matched standard curve could be obtained for the six analytes. The method detection limit was 0.1 μg g^?1 for melamine and cyromazine, 0.2 μg g^?1 for ammeline and ammelide, 1.2 μg g^?1 for cyanuric acid and 1.0 μg g^?1 for biuret in sediment matrix. The recoveries of these compounds were 70.1–98.3% and the relative standard deviations were 0.5–4.4%. Finally, the proposed method was successfully applied to the analysis of the sediment sample near the wastewater outlet of a melamine-producing factory.     

Determination of Melamine and Derivatives in Foods by Liquid Chromatography Coupled to Atmospheric Pressure Chemical Ionization Mass Spectrometry and Diode Array Detection

Two liquid chromatographic methods based on atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS) and diode array detection (DAD) are evaluated for the rapid determination of melamine (MEL) and structurally related compounds, including ammeline (AMN), ammelide (AMD), and cyanuric acid (CA) in foods. Both procedures used ion-exchange LC and isocratic elution. Samples were extracted by homogenization with acetonitrile/water/diethylamine. Specificity was demonstrated for LC-MS by the retention characteristics and MS spectra, by comparing with commercial standards. Specificity was only demonstrated in the case of LC-DAD for MEL and AMN, considering the retention characteristics and UV spectra. The recoveries obtained for spiked samples were satisfactory for all the analytes with LC-MS. The proposed procedure, LC-APCI-MS, was successfully applied to the analysis of different baby foods, including infant formula and breakfast cereal, and samples of rice flour, potato starch, soya drink, and coconut drink.     

Molecularly imprinted monolith coupled on-line with high performance liquid chromatography for simultaneous quantitative determination of cyromazine and melamine

We report a novel method for simultaneous determination of cyromazine and melamine based on a molecularly imprinted monolith on-line coupled with high performance liquid chromatography (HPLC). The imprinted monolith was prepared by in situ polymerization using 2,4-diamino-6-undecyl-1,3,5-triazine (DAUTA) as a mimic template. Due to the better solubility of DAUTA in chloroform, hydrogen bonds were effectively developed between the template and the functional monomer and resulted in the formation of highly specific cavities in the obtained imprinted monolith. With methanol as the loading solvent, cyromazine and melamine were both selectively retained by the obtained imprinted monolith, while the nonspecific adsorption on the non-imprinted monolith was negligible. The imprinted monolithic column was on-line coupled with HPLC for purification and concentration of the two analytes from milk samples. To minimize the peak broadening during the on-line transfer of the analytes from the imprinted monolith to the following analytical column, a successive desorption program was developed for the elution step, which enabled on-line stacking of the target compounds before being analyzed by HPLC. Low detection limits of 0.12 μg mL(-1) for melamine and 0.05 μg mL(-1) for cyromazine were achieved with only 0.3 mL of milk sample and a low sensitivity HPLC-UVD instrument. The method may be further extended to detect other analytes of interest in a large variety of samples.     

基质分散固相萃取-高效液相色谱-串联质谱法快速测定婴幼儿配方乳粉中19种非蛋白氮化合物

建立了基质分散固相萃取-高效液相色谱-串联质谱法同时检测婴幼儿配方乳粉中的三聚氰胺、灭蝇胺、脒脲、氨基三嗪、3-氨基三唑、4-氨基三唑、三聚氰酸、双氰胺、硫脲、氨基脲、L-亮氨酸、L-异亮氨酸、L-精氨酸、L-羟脯氨酸、三聚氰胺一酰胺、三聚氰胺二酰胺、胍、尿囊素、L-茶氨酸等19种非蛋白氮化合物的方法。样品用甲酸沉淀蛋白,三氯甲烷除去脂肪,乙腈提取,无水硫酸镁去除多余水分, C18净化,Merck ZIC HILIC 色谱柱(150 mm ×2.1 mm,5μm,200魡)分离,梯度洗脱,电喷雾离子化正离子模式(ESI+)和负离子模式( ESI-)同时扫描,多反应监测模式进行检测。其中,尿囊素采用外标法定量,其余18种化合物采用内标法定量。本方法对19种化合物的线性相关系数( r)均大于0.99,定量限( LOQ)为0.05~5.0 mg/kg,回收率为82.2%~115%, RSD<20%。     

Fast quantitative determination of melamine and its derivatives by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A simple, sensitive and fast method for the determination of melamine and its derivatives in milk powder using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was developed. Neither time-consuming sample preparation, nor special target plates, or other extra equipment are necessary. The common matrix sinapinic acid (SA) was used with a dried-droplet preparation. Detection limits (signal-to-noise (S/N) ratio = 3) for standard solutions of melamine, ammeline and cyanuric acid were 10, 25 and 10 μg/L, respectively. The limit of quantification (LOQ) for melamine was 25 μg/L and excellent linearity (R(2): 0.9990) was maintained over the range of 10-2000 μg/L. Ammeline and cyanuric acid were analyzed with an LOQ of 50 μg/L and also excellent linearity (R(2): 0.9997 and R(2): 0.9998). Good accuracy and precision were obtained for all concentrations within the range of the standard curve. The developed method was successfully used for the determination of melamine, ammeline and cyanuric acid in milk powder samples with a simple sample preparation. The LOQ of melamine was 0.5 μg/g. Ammeline and cyanuric acid were detectable at 0.5 and 5 μg/g. This method showed excellent accuracy, precision and linearity and significantly reduces the needed analysis time, as only approximately 10 s/sample measuring time is required. To the authors' knowledge, this is the first published method to quantify melamine and derivatives by MALDI-TOF-MS.     

亲水色谱-串联质谱法对牛奶与奶粉中三聚氰胺及其衍生物含量的测定

建立了牛奶和奶粉中三聚氰胺及其3种衍生物（三聚氰酸、三聚氰酸一酰胺、三聚氰酸二酰胺）的亲水色谱-串联质谱检测方法（HILIC-MS/MS）。牛奶样品直接用乙腈超声波提取;奶粉样品先用水溶解后再加乙腈超声波提取,高速离心后以乙腈-水为流动相,采用亲水作用色谱柱分离这4种化合物,在电喷雾正、负电离切换多反应监测模式下进行定性与定量分析,三聚氰酸一酰胺与三聚氰酸二酰胺含量在50～10000μg/kg之间、三聚氰胺与三聚氰酸在25～5000μg/kg之间,均获得良好的线性。方法检出限（LOD）为5～20μg/kg,定量下限（LOQ）为50～100μg/kg,3个添加水平的平均回收率为81%～97%。该方法简便、快速、准确,可满足牛奶及奶粉中三聚氰胺、三聚氰酸、三聚氰酸一酰胺、三聚氰酸二酰胺测定的需要。     

Quantitative Bestimmung von Melamin, Ammelin, Ammelid und Cyanurs?ure im quatern?ren Gemisch. II

Zusammenfassung Zur quantitativen Bestimmung von Melamin, Ammelin, Ammelid und CyanursÄure im quaternÄren Gemisch werden in einer Probe Melamin, Ammelin und Ammelid mit Hilfe eines Anionenaustauschers durch aufeinanderfolgende Eluierung mit Wasser, 0,5 bzw. 2 N NaOH-Lösungen getrennt und spektrophotometrisch in saurer Lösung bei 235, 230 und 215 nm bestimmt. CyanursÄure wird in einer zweiten Probe durch Zurückhalten der Cyanurbasen auf einem stark sauren Kationenaustauscher abgetrennt und als Melamincyanurat gravimetrisch bestimmt. Im Vergleich mit der früher von uns gegebenen chemischen Methode [6] ist die gegenwÄrtige vorteilhafter, da sie bei ungefÄhr gleicher Genauigkeit schneller und mit kleineren Substanzmengen zum Ziel führt.

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Quantitative determination of melamine, ammeline, ammelide and cyanuric acid in the quaternary mixture. II

A method for the quantitative determination of melamine, ammeline, ammelide and cyanuric acid in a quaternary mixture is proposed. In one sample melamine, ammeline and ammelide are separated from an anion-exchanger by succesive elution with water, 0.5 and 2 N NaOH solutions, respectively, and are determined spectrophotometrically in an acid solution at 235 nm, 230 nm and 215 nm. In a second sample, cyanuric acid is separated on a strongly acid cation-exchanger by retaining the cyanuric bases and is determined gravimetrically as melamine cyanurate. In comparison with the chemical method previously proposed [6] the present one is more advantageous since it offers the same accuracy, yet more rapidly, and requiring smaller samples.

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I. Mitteilung: G. Ostrogovich u. R. Bacaloglu [6].     

应用亲水作用色谱检测鸡蛋和肉中三聚氰胺及三聚氰酸二酰胺

应用亲水作用色谱(HILIC)对从市场上购买的鸡蛋和肉中的三聚氰胺及三聚氰胺降解产物三聚氰酸二酰胺进行了检测。采用的色谱柱为ZIC-HILIC柱,流动相为3 mmol/L磷酸二氢铵溶液(pH 6.9)-乙腈(20:80, v/v),流速为0.8 mL/min,检测波长为220 nm。在该体系下,三聚氰胺和三聚氰酸二酰胺的保留时间适中,与样品中的内源性物质有良好的分离。样品经0.1%磷酸提取,偏磷酸及乙腈沉淀蛋白质和糖类物质,以及P-SCX固相萃取柱净化。三聚氰胺和三聚氰酸二酰胺在0.4～40 mg/L范围内与峰面积呈良好的线性关系,样品定量限(按信噪比(S/N)不小于10计)为2 mg/kg,在2～10 mg/kg添加水平下的平均回收率为80%～105%,相对标准偏差小于10%。该方法具有良好的分离选择性,可用于鸡蛋和肉中三聚氰胺和三聚氰酸二酰胺的同时检测。     

Membrane sampling with microdialysis coupled to HPLC/UV for on-line simultaneous determination of melamine and cyanuric acid in non-dairy coffee creamer

An on-line microdialysis/high-performance liquid chromatography method was developed for the simultaneous determination of melamine and cyanuric acid in non-dairy coffee creamer. To collect these analytes from aqueous samples, the microdialysis system featured a microdialysis probe incorporating a polyarylethersulfone membrane and employed 0.05 M HCl in 0.1% (v/v) MeOH as the perfusate, with optimal efficiency obtained at a flow rate of 1 μL min(-1). The chromatographic conditions were optimized when using a reverse-phase phenyl column and a mobile phase of phosphate buffer solution in 10% (v/v) MeOH, buffered at pH 3.0. Good linearity relationship (r(2) > 0.9987), intra- and inter-day precisions (RSDs < 6.6%), recoveries (96.9 - 105.0%), and limits of detection (melamine, 3 ppb; cyanuric acid, 150 ppb) were observed for the two analytes. This method has been successfully applied to simultaneous determination of melamine and cyanuric acid in commercial creamers with the recoveries in the range of 97.5 to 102.6%.     

SPE then RP-LC for Simultaneous Analysis of Cyromazine and its Metabolite Melamine in Liquid Milk and Egg

Solid-phase extraction (SPE) and reversed-phase liquid chromatography (RP-LC) have been used for simple, sensitive simultaneous analysis of cyromazine and melamine residues in liquid milk and eggs. The conditions used for SPE and LC were investigated and optimized. A combined cation-exchange–reversed-phase cartridge was used for clean-up, and an ODS (C₁₈) column (150 mm × 4.6 mm i.d., 5-μm particles) with 62:38 (v/v) 5 mm sodium lauryl sulfate (pH 3.4)–acetonitrile as mobile phase was used for RP-LC. Under the optimum conditions the method limit of detection (LOD) for both cyromazine and melamine was 6.2 μg kg^?1 for liquid milk samples, and 11.5 μg kg^?1 for egg samples. Average recovery of cyromazine and melamine from milk samples was 90.3%, RSD 4.6–5.6%, and 99.6%, RSD 3.2–4.7%, respectively. Average recovery of cyromazine and melamine from egg samples was 85.3%, RSD 1.0–4.7%, and 89.6%, RSD 3.1–5.0%, respectively. The method enables detection of melamine and cyromazine at levels as low as 20.7 μg kg^?1 in liquid milk and 38.3 μg kg^?1 in egg.     

SPE then RP-LC for Simultaneous Analysis of Cyromazine and its Metabolite Melamine in Liquid Milk and Egg

Solid-phase extraction (SPE) and reversed-phase liquid chromatography (RP-LC) have been used for simple, sensitive simultaneous analysis of cyromazine and melamine residues in liquid milk and eggs. The conditions used for SPE and LC were investigated and optimized. A combined cation-exchange–reversed-phase cartridge was used for clean-up, and an ODS (C₁₈) column (150 mm × 4.6 mm i.d., 5-μm particles) with 62:38 (v/v) 5 mm sodium lauryl sulfate (pH 3.4)–acetonitrile as mobile phase was used for RP-LC. Under the optimum conditions the method limit of detection (LOD) for both cyromazine and melamine was 6.2 μg kg⁻¹ for liquid milk samples, and 11.5 μg kg⁻¹ for egg samples. Average recovery of cyromazine and melamine from milk samples was 90.3%, RSD 4.6–5.6%, and 99.6%, RSD 3.2–4.7%, respectively. Average recovery of cyromazine and melamine from egg samples was 85.3%, RSD 1.0–4.7%, and 89.6%, RSD 3.1–5.0%, respectively. The method enables detection of melamine and cyromazine at levels as low as 20.7 μg kg⁻¹ in liquid milk and 38.3 μg kg⁻¹ in egg.

     

Development of a pressurised liquid extraction and liquid chromatography with electrospray ionization-tandem mass spectrometry method for the determination of domoic acid in shellfish

Amnesic shellfish poisoning (ASP) is a potentially lethal human toxic syndrome which is caused by domoic acid (DA) that originates in marine phytoplankton belonging to the Pseudonitzschia genus. A confirmatory and sensitive procedure has been developed and validated for the determination of DA in shellfish. The proposed method includes pressurised liquid extraction (PLE) with methanol/acetone (9:1), florisil clean-up purification inside the PLE extraction cell and detection by liquid chromatography (LC) coupled to electrospray ionization in positive mode tandem mass spectrometry (ESI-MS-MS). Comparison of ionization sources (ESI, atmospheric pressure ionization (APCI) atmospheric pressure photoionization (APPI) and combined APCI/APPI) were carried out in order to improve the analytical signal. The main parameters affecting the performance of the different ionization sources and PLE parameters were previously optimised using statistical design of experiments (DOE). Linear calibrations were obtained using mussel tissue extracts 0.05-5 microg DA/ml (R2>0.999). The limits of detection (LOD) and quantitation (LOQ) of the method were 0.2 and 0.5 microg/g respectively and recoveries ranged from 81 to 95%. This method was successfully applied to determine DA levels in 46 shellfish samples collected from Valencian (Spain) supermarkets, showing high sample throughput.     

Dabsyl chloride derivatisation of melamine followed by high-performance liquid chromatography determination in water samples

A new and sensitive precolumn derivatisation with dabsyl chloride was developed for the analysis of melamine in water samples by high-performance liquid chromatography (HPLC) with visible detection. Derivatisation with dabsyl chloride leads to improving sensitivity and hydrophobicity of melamine. Under optimum conditions of derivatisation and microextraction, the method yielded a linear calibration curve ranging from 10 to 2000 µg L^?1 with a determination coefficient (R²) of 0.9952. Limit of detection (LOD) and limit of quantification (LOQ) were 2.0 and 6.0 µg L^?1, respectively. The relative standard deviation per cent (RSD%) for intraday and inter-day extraction and determination at 20 and 200 µg L^?1 levels of melamine was less than 8.2% (n = 6). Finally, the proposed method was successfully applied for the determination of melamine in different water samples and satisfactory results were obtained (relative recovery ≥91%).     

Method development and validation for melamine and its derivatives in rice concentrates by liquid chromatography. Application to animal feed samples

An isocratic LC method for the determination of melamine and its degradation products (ammelide, ammeline, and cyanuric acid), used to increase the apparent protein content of rice protein concentrate, has been developed. Method development involved optimization of different RP columns, aqueous mobile phases, pH, phosphate concentration, and temperature. The optimum separation of these compounds was achieved using a Luna CN column (30 °C), 5 mmol L⁻¹ sodium phosphate (pH 5.0) as mobile phase, 1 mL min⁻¹ flow-rate, UV absorbance-DAD detection at 220 nm, and resorcine as internal standard; this enabled separation of these compounds with baseline resolution (values in the 2.1–10.1 range) in about 8 min. Prior to HPLC, the developed sample preparation procedure consisted in a leaching process using the above mentioned mobile phase. Method validation was carried out in rice protein concentrates in accordance with the European Commission decision 2002/657/EC criteria. For this purpose, eight mandatory performance characteristics for the conventional validation approach were determined: calibration graphs, extraction efficiencies, decision limits, detection capabilities, precision (repeatability and within-laboratory reproducibility), accuracy, selectivity, and robustness. The extraction efficiencies for these compounds were in the range 99–100% and the within-laboratory reproducibility at 1.0, 1.5, and 2.0 detection capabilities concentration levels were smaller than 5, 4, and 3%, respectively. Finally, the proposed method was successfully applied to the analysis of other rice protein concentrates and several animal feed samples.     

Highly sensitive detection of melamine and its derivatives by capillary electrophoresis coupled with online preconcentration techniques

Two kinds of online preconcentration techniques including large-volume sample stacking-sweeping and selective-exhaustive injection-sweeping (SEI-S) were employed in CE to develop a rapid, simple and sensitive method for determination of melamine (MM) and its derivatives such as ammeline (AMN), ammelide (AMD) and cyanuric acid (CA) in liquid milk products. Though the sensitivity of large-volume sample stacking-sweeping is not particularly high, it can be used to detect all the four compounds simultaneously. However, it is very difficult to improve the sensitivity of these four compounds by using single SEI-S method directly, owing to their completely different extents of protonation or deprotonation. Grouping can solve this problem perfectly. After the four compounds are divided into two groups (MM/AMN and AMD/CA), cation-SEI-S or anion-SEI-S can be applied for their online stacking. In cation-SEI-S, the detection limits for MM and AMN were both 0.01 ng/mL based on the signal-to-noise ratio of 3. In anion-SEI-S, the detection limits were 0.05 and 0.02 ng/mL for CA and AMD, respectively. Results of this study show a great potential for the SEI-S method to be a useful tool for the rapid and sensitive determination of MM and its derivatives in liquid milk products.     

Validation of a high-performance chromatographic method for determination of cefotaxime in biological samples

An analytical method for detecting and quantifying cefotaxime in plasma and several tissues is described. The method was developed and validated using plasma and tissues of rats. The samples were analyzed by reversed phase liquid chromatography (HPLC) with UV detection (254 nm). Calibration graphs showed a linear correlation (r > 0.999) over the concentration ranges of 0.5–200 μg/mL and 1.25–25 μg/g for plasma and tissues, respectively. The recovery of cefotaxime from plasma standards prepared at the concentrations of 25 μg/mL and 100 μg/mL was 98.5 ± 3.5% and 101.8 ± 2.2%, respectively. The recovery of cefotaxime from tissue standards of liver, fat and muscle, prepared at the concentration of 10 μg/g was: 89.8 ± 1.2% (liver), 103.9 ± 6.5% (fat) and 97.8 ± 2.1% (muscle). The detection (LOD) and quantitation (LOQ) limits for plasma samples were established at 0.11 μg/mL and 0.49 μg/mL, respectively. The values of these limits for tissues samples were approximately 2.5 times higher: 0.3 μg/g (LOD) and 1.25 μg/g (LOQ). For plasma samples, the deviation of the observed concentration from the nominal concentration was less than 5% and the coefficient of variation for within-day and between-day assays was less than 6% and 12%, respectively. The method was used in a pharmacokinetic study of cefotaxime in the rat and the mean values of the pharmacokinetic parameters are given.     

Preparation of a magnetic molecularly imprinted polymer with pseudo template for rapid simultaneous determination of cyromazine and melamine in bio-matrix samples

A magnetic molecularly imprinted polymer (M-MIP) for cyromazine and melamine was prepared by simple suspension polymerization using a pseudo template, 2-(4,6-diamino-1,3,5-triazin-2-ylamino)ethanethiol disulfide. The M-MIP was characterized by scanning electron microscopy, Fourier-transform infrared spectroscopy, and vibrating sample magnetometry. Molecular recognition properties and binding capability to cyromazine and melamine were evaluated by adsorption testing, which showed the M-MIP had better affinity and selectivity than the magnetic non-imprinted polymer (M-NIP) for cyromazine and melamine. A method based on molecularly imprinted solid-phase extraction assisted by magnetic separation was developed for extraction of cyromazine and melamine from bio-matrix samples. Various conditions, for example desorption conditions, amount of M-MIP, extraction time, and sample pH were optimized. High-performance liquid chromatography with UV detection was used to determine cyromazine and melamine after extraction. The proposed method was successfully applied to determination of cyromazine and melamine in egg and milk samples. Recovery of standard spiked cyromazine and melamine from these samples was between 71.86 and 80.57%, with intraday and interday relative standard deviation ranging from 3.45 to 6.39% and from 3.95 to 7.84%, respectively. The results indicate that the pseudo template M-MIP can be used for preconcentration, purification, and analysis of cyromazine and melamine in bio-matrix samples.     

Analysis of Melamine,Cyanuric Acid,Ammelide, and Ammeline Using Matrix‐Assisted Laser Desorption Ionization/Time‐of‐Flight Mass Spectrometry (MALDI/TOFMS)

Abstract

Melamine, cyanuric acid, two compounds connected to tainted pet food, and related analogs have been analyzed using matrix‐assisted laser desorption ionization/time‐of‐flight mass spectrometry. (M+H)⁺ ions were observed for ammelide and ammeline under positive ion conditions with sinapinic acid as the matrix. With alpha‐cyano‐4‐hydroxy‐cinnamic acid as the matrix, a matrix‐melamine complex was observed; however, no complex was observed with sinapinic acid as the matrix. (M?H)^? was observed for cyanuric acid with sinapinic acid as the matrix.     

Determination of melamine and related triazine by-products ammeline, ammelide, and cyanuric acid by micellar electrokinetic chromatography

In this study, micellar electrokinetic chromatographic (MEKC) methods were developed for the detection of traces of melamine and its related by-products (ammeline, ammelide, and cyanuric acid). Two on-line sample concentration steps namely reversed electrode polarity stacking mode (REPSM) and cation-selective injection (CSI) were used for improving the detection sensitivity. For REPSM, a borate-NaOH buffer (pH 10, 35 mM) composed of 60 mM SDS and 10% (v/v) methanol, was used as carrier electrolyte, and samples were prepared in an aqueous solution of 10 mM NaOH. In CSI, a phosphate buffer (pH 2, 50 mM) containing 41 mM SDS was used as the carrier electrolyte, and samples were prepared with an aqueous solution of 10 mM NaOH and a phosphate buffer (pH 2.0, 25 mM) in a volume ratio of 1:9. The results indicated that REPSM enhanced all analyte signals except for melamine, which could be concentrated only by the CSI. The detection limit was reduced from 1.7 mg L⁻¹ to 2.8 μg L⁻¹ for melamine by the optimal CSI step, and from 0.23-1.2 mg L⁻¹ to 2.4-5.0 μg L⁻¹ for the other three analytes by the optimal REPSM step. Tableware made of melamine and samples of flour were used as test samples, and the results indicated that the proposed MEKC methods can successfully determine contaminations from melamine. The study also indicated that when the plastic made of melamine was exposed only once to an acidic solution (acetic or phosphoric acid) at 80 °C for 30 min, melamine continuously leached out from the test sample even without any further treatment with an acidic solution.     

Determination of anti‐malaria agent chloroquine using single drop liquid‐liquid‐liquid microextraction

A simple, sensitive, and inexpensive single drop liquid‐liquid‐liquid microextraction combined with isocratic RP‐HPLC and UV detection was developed for the determination of anti‐malaria drug, chloroquine. The target compound was extracted from alkaline aqueous sample solution (adjusted to 0.5 mol/L sodium hydroxide) through a thin layer of organic solvent membrane and back‐extracted to an acidic acceptor drop (adjusted to 0.02 mol/L phosphoric acid) suspended on the tip of a 25 μL HPLC syringe in the organic layer. This syringe was also used for direct injection after extraction. The linear range was 1–200 μg/L. The LOD and LOQ were 0.3 and 1.0 μg/L, respectively. Intra‐and inter‐day precisions were less than 2.0 and 2.3%, respectively. The real samples were successfully analyzed using the proposed method. The recoveries of spiked samples were more than 94.6%.