Determination of multi-residue for malachite green,gentian violet and their metabolites in aquatic products by high-performance liquid chromatography coupled with molecularly imprinted solid-phase extraction

A sorbent was synthesized and investigated for molecularly imprinted solid-phase extraction (MISPE). Molecularly imprinted polymers (MIPs) were synthesized via precipitation polymerization procedure, where methacrylic acid (MAA) was used as functional monomer and ethylene glycol dimethacrylate (EDMA) as cross-linking agent. The imprinting effect and selectivity of the MISPE were evaluated by elution experiments. The resulting MISPE showed high extraction selectivity to malachite green, gentian violet and their metabolites, which may be caused by both the ion exchange and the hydrophobic interactions. The determination of multi-residue for malachite green, gentian violet and their metabolites in aquatic products by HPLC coupled with MISPE was also investigated. The mean recoveries calculated by solvent calibration curve for malachite green (MG), gentian violet (GV), leucomalachite green (LMG) and leucogentian violet (LGV) were from 89.8% to 99.1% for grass carp, 90.6% to 101.2% for shrimp and 91.3% to 96.3% for shellfish. The decision limit (CCα) and the detection capability (CCβ) obtained for MG, GV, LMG and LGV were in the range of 0.11–0.14 and 0.19–0.24 μg kg⁻¹ for grass carp, shrimp and shellfish. The MISPE was successfully used off-line for the determination of MG, GV and their metabolites in aquatic products.     

Confirmatory analysis of malachite green, leucomalachite green, crystal violet and leucocrystal violet in salmon by liquid chromatography-tandem mass spectrometry

A method has been developed to analyse for malachite green (MG), leucomalachite green (LMG), crystal violet (CV) and leucocrystal violet (LCV) residues in salmon. Salmon samples were extracted with acetonitrile:McIIIvain pH 3 buffer (90:10 v/v), sample extracts were purified on a Bakerbond strong cation exchange solid phase extraction cartridge. Aliquots of the extracts were analysed by LC-MS/MS. The method was validated in salmon, according to the criteria defined in Commission Decision 2002/657/EC. The decision limit (CCalpha) was 0.17, 0.15, 0.35 and 0.17 microg kg(-1), respectively, for MG, LMG, CV and LCV and for the detection capability (CCbeta) values of 0.30, 0.35, 0.80 and 0.32 microg kg(-1), respectively, were obtained. Fortifying salmon samples (n=6) in three separate assays, show the accuracy to be between 77 and 113% for MG, LMG, LCV and CV. The precision of the method, expressed as RSD values for the within-laboratory reproducibility, for MG, LMG and LCV at the three levels of fortification (1, 1.5 and 2.0 microg kg(-1)), was less than 13%. For CV a more variable precision was obtained, with RSD values ranging between 20 and 25%.     

Ultrasonic enhancement of leaching and in situ derivatization of haloacetic acids in vegetable foods prior to gas chromatography-electron capture detection

With a new oxidant for post-column chemical derivation, a novel approach was developed for the determination of Malachite Green (MG) and Leucomalachite Green (LMG) in fish by high-performance liquid chromatography (HPLC). A C(8) column was used for separation, and elution was performed with a pH 2.5 phosphate buffer (0.02molL(-1)) containing 40% acetonitrile. When the eluate was combined with 3.0x10(-4)molL(-1) iodine solution, LMG was converted to MG and detected at 618nm after post-column derivatization. The recoveries of MG and LMG were ranged from 67.3% to 73.9% and 84.7% to 92.1%, respectively, which were obtained by measuring the amount of MG and LMG in the samples with solvent calibration curve. The decision limit (CCalpha) and the detection capability (CCbeta) obtained for MG and LMG were in the range of 0.10-0.17 and 0.13-0.23mugkg(-1) in grass carp, shrimp and shellfish. This method appeared suitable for the control of MG and LMG residues in aquatic products.     

免标记比色核酸适配体传感器同步快速检测孔雀石绿和无色孔雀石绿

研究以双特异性核酸适配体A3作为传感探针、纳米金（AuNPs）为指示剂、NaCl溶液为聚集诱导剂,构建了一种新型的免标记AuNPs比色生物传感器,可实现水产品中孔雀石绿（MG）和无色孔雀石绿（LMG）的同步、快速、可视化检测。该方法的检测原理是核酸适配体A3对MG和LMG有双特异性识别能力,可作为MG和LMG理想的识别受体。它可通过静电作用吸附到AuNPs表面,保护AuNPs并抑制高盐溶液诱导的聚集,AuNPs溶液颜色不变,即为红色;当加入靶标MG或LMG后,该核酸适配体能够与靶标特异性结合,并从AuNPs表面上解离,AuNPs失去保护作用而在高盐溶液诱导下发生聚集,溶液颜色由红变蓝。根据颜色变化,可通过肉眼定性或通过光谱仪定量分析MG和LMG的残留量。该方法首先将50 μL的核酸适配体A3（终浓度150 nmol/L）与150 μL的AuNPs（终浓度1.25 nmol/L）混合,室温孵育6 min。随后加入50 μL待测液,室温孵育30 min。最后加入50 μL NaCl（终浓度150 mmol/L）,4 min后观察溶液颜色变化,并分别测定MG和LMG在520 nm和650 nm下的吸光度值。结果表明,在最佳反应条件下,该方法能够特异性检测MG和LMG,而对磺胺嘧啶（SDZ）和硝基呋喃妥因（NFT）无交叉反应;当MG、LMG的浓度为0~17.5 μmol/L时,吸光度比值与靶标浓度呈现良好的线性关系,相关系数（R ² ）分别为0.9938和0.9715。MG和LMG的检出限分别为6.93 nmol/L和6.38 nmol/L,加标回收率分别为88.60%~93.30%和101.80%~107.00%,相对标准偏差（RSD）分别为2.27%~3.55%和2.62%~3.75%。该方法操作简单,快速和灵敏,可为水产品中MG和LMG的同步快速检测提供一种新方法。     

Determination of the sum of malachite green and leucomalachite green in salmon muscle by liquid chromatography-atmospheric pressure chemical ionisation-mass spectrometry

A sensitive method for the determination and confirmation of the sum of malachite green (MG) and leucomalachite green (LMG) in salmon muscle has been developed. It is based on the use of an oxidative pre-column reaction which converts LMG into MG previous to liquid chromatography-atmospheric pressure chemical ionisation-mass spectrometry (LC-APCI-MS) analysis. The determination of both compounds together constitutes a good screening method to confirm the presence of this kind of residue, taking into account that the combined signals will provide a gain of sensitivity. The detection limit, determined for spiked salmon samples using the confirmatory ion m/z 313, was 0.15 microg/kg. The recoveries determined at a spiking level of 2 microg/kg were 85 and 70% for LMG and MG, respectively, with respective relative standard deviations of 1.3 and 3.1%.     

Advantages of PARAFAC calibration in the determination of malachite green and its metabolite in fish by liquid chromatography-tandem mass spectrometry

This paper reports the properties and advantages of the three-way calibration models based on parallel factor analysis (PARAFAC) in the simultaneous determination of malachite green (MG) and its metabolite (leucomalachite green, LMG) in trout. A recently method proposed by community reference laboratory AFSSA-LERMVD (Fougères, France) has been used. The method is based on liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS) in multiple reaction monitoring (MRM) mode. The validation of the method has been carried out taking into account the Decision 2002/657/EC. The figures of merit for PARAFAC and univariate calibration models of six non-consecutive days analyzed during a month were evaluated. With the samples of the first 3 days, calibration models were built and the fish fortification samples of the other days were predicted. Decision limits (CCalpha, alpha=0.01), detection capabilities (CCbeta, beta=0.05) and mean relative errors in absolute value (in calibration and with test samples) obtained with PARAFAC calibrations were more homogeneous than the ones obtained with the univariate calibrations, especially in LMG. These figures of merit were in the range of 0.2-0.83 microg kg(-1) (CCbeta) and 0.2-0.49 microg kg(-1) (CCalpha), whereas mean relative errors in absolute value were in the range of 1.1-7.4% in calibration and 3-12% in test samples for MG and LMG with PARAFAC calibrations. The PARAFAC calibrations allow detecting the test samples which are not similar to the calibration samples and in this way their wrong quantification is avoided.     

液相色谱-串联质谱法同时测定水产品中孔雀石绿、 结晶紫以及它们的隐色代谢物残留

采用液相色谱-串联质谱法(LC-MS/MS)同时测定水产品中的孔雀石绿、结晶紫以及它们的隐色代谢物残留。匀质后的水产品样品用乙腈和乙酸铵缓冲液提取。合并提取液,用二氯甲烷反提取,经中性氧化铝柱和PRS柱固相萃取净化。采用ZORBAX SB-C18色谱柱,并以0.5 mmol/L乙酸铵-乙腈(体积比为10∶90)混合溶液为流动相,无需使用氧化铅柱在线氧化,色谱分离后直接进入串联质谱检测器检测。采用电喷雾离子源,正离子多反应监测(MRM)模式检测。方法的检测限（S/N=3）可达0.5 ng/g,平均加标回收率为77.6%～98.1%,相对标准偏差均小于8.2%。大量实际水产品样品的检测结果表明,此方法适合于对水产品中孔雀石绿、结晶紫以及它们的隐色代谢物的残留检测。     

Determination of malachite green and leucomalachite green in a variety of aquacultured products by liquid chromatography with tandem mass spectrometry detection

A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for determining the residues of malachite green (MG) and leucomalachite green (LMG) in a number of aquatic species. MG and its metabolite were extracted from homogenized tissues with a perchloric acid-acetonitrile solution; the extract was centrifuged; and an aliquot was taken, concentrated, and passed through a chemically bonded octadecyl C18 solid-phase extraction column. The compounds of interest were eluted with acetonitrile, and the eluate was evaporated to dryness. The residue was dissolved in acetonitrile and diluted with water in preparation for analysis by LC/MS/MS. MG and its metabolite were determined by reversed-phase LC using a Luna C18 column with an ammonium hydroxide-formic acid buffer in acetonitrile gradient and MS/MS detection using multiple reaction monitoring. Calibration curves were linear for all analyses between 5 and 500 pg injected for both analytes, with recoveries ranging from 81% for LMG to 98% for MG in salmon spiked at the 1 ng/g level. Detection limits of 0.1 ng/g for both MG and LMG were easily obtainable using the recommended method. The operational errors, interferences, and recoveries for spiked samples compared favorably with those obtained by established methodology. The recommended method is simple, rapid, and specific for monitoring residues of MG and LMG in a number of aquatic species.     

Determination and confirmation of malachite green and leucomalachite green residues in salmon using liquid chromatography/mass spectrometry with no-discharge atmospheric pressure chemical ionization

A liquid chromatography/mass spectrometry (LC/MS) method was developed to quantitate and confirm residues of leucomalachite green (LMG) in salmon tissue after their conversion to chromic malachite green (MG) in the extraction process. The method uses no-discharge atmospheric pressure chemical ionization (APCI) in conjunction with an ion-trap instrument to generate product-ion spectra. In the sample preparation procedure, salmon tissue is extracted with acetonitrile/buffer, the LMG residue is partitioned into methylene chloride, the LMG is converted to MG using an organic oxidizing agent, and the MG is isolated on alumina/propylsulfonic acid solid-phase extraction cartridges. The method was validated by fortifying salmon with different levels of LMG, and then detecting the residue as MG The LC/MS conditions, including a comparison of electrospray and no-discharge APCI, were evaluated and optimized. MG was not confirmed in any of the control tissue extracts, and all fortified samples analyzed during validation met the confirmation criteria as described. In addition to providing confirmatory data, this method can provide an alternative method for quantitation of MG in salmon. The recoveries of LMG measured as MG by this LC/MS method, at fortification levels of 1-10 ng/g were very high (86-109%), with low relative standard deviation(RSD) values (6.4-13%). The results agreed very closely with those obtained for the same extracts using an LCNIS procedure, indicating that matrix suppression was not an issue. The presence of LMG in salmon tissue samples fortified at 0.25 ng/g was confirmed by this method, with an average recovery of 70.1% and an RSD of 12.0%. Sample extracts from fish exposed to MG were also analyzed.     

Liquid chromatographic determination of malachite green and leucomalachite green (LMG) residues in salmon with in situ LMG oxidation

A liquid chromatography (LC) method is presented for the quantitative determination of malachite green (MG) in salmon. MG and leucomalachite green (LMG) residues were extracted from salmon tissue with ammonium acetate buffer and acetonitrile, and then isolated by partitioning into dichloromethane. LMG was quantitatively oxidized to the chromic MG by reaction with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone. Samples were then cleaned up by solid-phase extraction with alumina and propylsulfonic acid phases. Extracts were analyzed for MG by LC with visible detection at 618 nm using isocratic elution and a C18 column. The method was validated in 35 farm-raised salmon (Salmo salar) tissues fortified at 1, 2, 4, and 10 ng/g (ppb) with an average recovery of 95.4% and a relative standard deviation of +/- 11.1%, and in 5 canned salmon (Oncorhynchus gorbuscha) samples fortified at 10 ng/g with an average recovery of 88.9 +/- 2.6%. This study also included the determination of MG and LMG residues in tissues from salmon that had been treated with MG MG was quantitatively determined at the method detection limit of 1 ng/g.     

超高效液相色谱-串联质谱法测定水产品中孔雀石绿、结晶紫及其代谢物残留量

采用超高效液相色谱-串联质谱法同时检测水产品中孔雀石绿、结晶紫及其代谢物(隐色孔雀石绿、隐色结晶紫)。经匀浆处理的水产品样品,用乙腈提取,加入酸性氧化铝去除油脂,旋转蒸发器蒸干后,用甲酸-乙腈-水(0.1+10+89.9)溶液溶解,样品溶液用超高效液相色谱分离,电喷雾串联四极杆质谱进行检测。以氘代孔雀石绿、氘代隐色孔雀石绿为内标物。孔雀石绿、结晶紫及其代谢物的质量浓度均在5.0μg·L-1以内与其峰面积呈线性关系,检出限(3S/N)在0.10～0.12μg.kg-1之间。以空白水产品样品为基体进行回收试验,方法的回收率在90.2%～108.0%之间,相对标准偏差(n=6)在2.3%～7.6%之间。     

超高效液相色谱-串联质谱法检测畜产品和水产品中3-甲基喹恶啉-2-羧酸残留

以猪肉、猪肝、猪肾、胖头鱼、对虾和蟹为试验材料,建立了喹乙醇(OLA)的残留标示物3-甲基喹恶啉-2-羧酸(MQCA)残留的超高效液相色谱-串联质谱(UPLC-MS/MS)检测方法。采用0.2 mol/L盐酸提取可食性组织中的分析物,经C18固相萃取小柱净化、35 ℃氮气吹干及含0.1%(v/v)甲酸的乙腈溶解后,采用超高效液相色谱分离,串联质谱法确证和定量分析。质谱检测采取正离子多反应监测模式,外标法定量。结果表明: MQCA在2～500 μg/L范围内呈良好的线性关系,各组织中的相关系数(r2)均大于0.990;猪肉、猪肝、猪肾、鱼、对虾和蟹中MQCA的检出限依次为0.90、1.51、0.94、1.04、1.62和1.80 μg/kg,定量限依次为3.00、5.02、3.13、3.46、5.40、6.00 μg/kg。从3～100 μg/kg的添加浓度的检测结果可以看出,MQCA的平均回收率均在73.6%与89.0%之间,日内相对标准偏差(RSD, n=5)在15%以下,日间RSD(n=3)为20%以下。该方法的灵敏度、准确度和精密度均符合兽药残留分析技术的要求,适用于动物组织中MQCA残留的定量分析和确证检测。     

Liquid chromatography with time-of-flight mass spectrometry for simultaneous determination of chemotherapeutant residues in salmon

Liquid chromatography with time-of-flight mass spectrometry (LC-TOF-MS) method has been developed for simultaneous confirmation by accurate mass measurement and quantitative determination of antibiotics (enrofloxacin, oxolinic acid, flumequine, erythromycin), fungicides (malachite green MG, leucomalachite green LMG) and parasiticide (emamectin benzoate) residues in edible portion of salmon. Confirmation of chemotherapeutant residues has been based on the system of identification points (IPs) established in the Commission Decision 2002/657/EC concerning the use of mass spectrometry (MS) techniques. A validation study on matrix is presented evaluating accuracy in terms of precision (λ_ppm 0.83-1.15) and trueness (0.22-0.70 Da). Limits of detection (LODs) and limits of quantification (LOQs) were in ranges of 1-3 and 3-9 μg/kg, below the maximum residue limits (MRLs) established in current EU legislation (100-200 μg/kg) for these chemotherapeutants. Considering the EU guidelines, decision limits (CCα) and detection capabilities (CCβ) were determined (ranges of 103-218 and 107-234 μg/kg, respectively) for authorised substances. For no authorised compounds (MG and LMG), LODs were 2 and 1 μg/kg, respectively, but exceed the MRPL (minimum required performance limit) established in the legislation which corresponds to the sum of MG and LMG (2 μg/kg). Acceptable intra-day and inter-day variability, in terms of relative standard deviation (R.S.D.) of the analytical method, were obtained (2-15%). Linearity was demonstrated from the LOQs of the analytes to 600 μg/kg (r > 0.9991). The method has involved an extraction procedure based on solid-liquid extraction (SLE) with recoveries higher than 80% for most target chemotherapeutants, with exception of enrofloxacin (40%).     

Determination of organotins in aquatic food by gas chromatography with pulsed flame photometric detection

A method based on gas chromatography (GC)-pulsed flame photometric detection (PFPD) was developed to determine the levels of organotins in aquatic food. After being purified by gel-permeation chromatography in ethyl actate-tetrahydrofuran, the organotin compounds were derivatized by pentylmagnesium bromide. The derivative products were injected into the GC system and detected by PFPD (sulfur mode). The method was validated by analysis of the certified reference material and spiked samples. Recoveries of organotins ranged from 84.1 to 116.6% with relative standard deviation between 1.3 and 16.0% when spiked at levels of 2, 10, and 40 microg/kg. The limits of detection varied from 0.1 to 1.2 microg/kg for shellfish and 0.1 to 0.5 microg/kg for fish. The proposed method was suitable for determining organotins in aquatic foods.     

A simple method for the determination of malachite green and leucomalachite green residues in fish by a modified QuEChERS extraction and LC/MS/MS

A simple method using LC/MS/MS was developed and validated to determine residues of malachite green (MG) and leucomalachite green (LMG) in fish fillet. A modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) technique was used to perform the sample preparation. The optimal extraction and cleanup conditions were established using an experimental design. The validation parameters obtained to determine both MG and LMG complied with the requirements established by regulatory agencies for the presence of such substances in fish, which establish that the method must attain, at least, a minimum required performance limit of 2 ng/g. The accuracy values ranged between 95 and 107%, and the precision values were lower than 11.2%. The method was used in the analysis of tilapia samples (n = 20) commercialized in Campinas, SP, Brazil. None of the samples presented detectable levels of MG or LMG residues.     

The effects of cooking on residues of malachite green and leucomalachite green in carp muscles

The effects of various cooking methods (boiling, baking and microwaving) on residues of malachite green (MG) and its major metabolite, leucomalachite green (LMG), in incurred carp muscles were investigated. Moreover, the stability of MG and LMG standard solutions under boiling in water and in oil was examined. The MG and LMG residues in cooked meat were determined by liquid chromatography with visible and fluorescence detectors. The results showed that in muscles cooked by boiling or baking MG concentration was reduced by 54% in 15 min while LMG was stable in these conditions. By microwave cooking MG residues were reduced by 61% after 1 min. Microwaving was the only method of cooking when a loss of LMG was observed (40% in 1 min). Both MG and LMG standard solutions were stable in boiling water at 100 degrees C. In cooking oil, MG was reduced by 49% after 10 min and less than 3% of the original MG remains after 90 min at 150 degrees C. No losses of LMG were observed over a time period of 120 min in cooking oil at 150 degrees C. Upon increasing the temperature to 210 degrees C and holding for 120 min, MG was rapidly reduced by 97% after 10 min. LMG under the same conditions was reduced by 18% after 10 min. No further loses of MG and LMG were observed after 120 min. The findings of this investigation show that the high temperature does not guarantee a full breakdown of residue of MG and LMG which may occur in carp muscles.     

Development and validation of a fast monoclonal based disequilibrium enzyme-linked immunosorbent assay for the detection of triphenylmethane dyes and their metabolites in fish

Malachite Green (MG), Crystal Violet (CV) and Brilliant Green (BG) are antibacterial, antifungal and antiparasitic agents that have been used for treatment and prevention of diseases in fish. These dyes are metabolized into reduced leuco forms (LMG, LCV, LBG) that can be present in fish muscles for a long period. Due to the carcinogenic properties they are banned for use in fish for human consumption in many countries including the European Union and the United States. HPLC and LC-MS techniques are generally used for the detection of these compounds and their metabolites in fish. This study presents the development of a fast enzyme-linked immunosorbent assay (ELISA) method as an alternative for screening purposes. A first monoclonal cell line producing antibodies to MG was generated using a hybridoma technique. The antibody had good cross-reactivates with related chromatic forms of triphenylmethane dyes such as CV, BG, Methyl Green, Methyl Violet and Victoria Blue R. The monoclonal antibody (mAb) was used to develop a fast (20 min) disequilibrium ELISA screening method for the detection of triphenylmethanes in fish. By introducing an oxidation step with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) during sample extraction the assay was also used to detect the presence of the reduced metabolites of triphenylmethanes. The detection capability of the assay was 1 ng g(-1) for MG, LMG, CV, LCV and BG which was below the minimum required performance limit (MRPL) for the detection method of total MG (sum of MG and LMG) set by the Commission Decision 2004/25/EC (2 ng g(-1)). The mean recoveries for fish samples spiked at 0.5 MRPL and MRPL levels with MG and LMG were between 74.9 and 117.0% and inter- and intra-assay coefficients of variation between 4.7 and 25.7%. The validated method allows the analysis of a batch of 20 samples in two to three hours. Additionally, this procedure is substantially faster than other ELISA methods developed for MG/LMG thus far. The stable and efficient monoclonal cell line obtained is an unlimited source of sensitive and specific antibody to MG and other triphenylmethanes.     

高效液相色谱串联质谱法检测腰果中黄曲霉毒素

建立了腰果中4种黄曲霉毒素的高效液相色谱-串联质谱检测方法(HPLC-MS/MS)。样品用甲醇-水(8:2, v/v)溶液提取后用弗罗里硅土柱净化,5 mL丙酮-水-甲酸溶液(96:3.5:0.5, v/v/v)洗脱,氮吹至干,1 mL甲醇定容;在资生堂MG C18色谱柱(100 mm×3.0 mm, 3 μm)上梯度洗脱分离,然后采用电喷雾离子化三重四极杆串联质谱测定。实验结果表明,4种黄曲霉毒素在各自的线性范围内峰面积与其质量浓度线性关系良好,相关系数(r2)大于0.997;检出限(信噪比为3)为0.009～0.04 μg/kg,定量限(信噪比为10)为0.03～0.12 μg/kg;平均回收率为63.0%～78.5%,相对标准偏差为2.8%～9.1%,均符合痕量分析的要求。评价了基质效应,信号抑制/增强值为88.8%～99.4%,说明净化后的基质效应较小。该方法简单快速、准确可靠,可用于腰果中黄曲霉毒素的检测。     

Production of malachite green oxalate and leucomalachite green reference materials certified for purity

Malachite green oxalate (MG oxalate) and leucomalachite green (LMG) have been prepared and certified as pure reference materials. The purities of MG oxalate and LMG were assessed by high-performance liquid chromatography–diode array detection (HPLC–DAD), nuclear magnetic resonance (NMR) spectroscopy, differential scanning calorimetry (DSC), Karl Fischer titration, ashing and thermogravimetric analysis (TGA). MG oxalate was purified by supercritical fluid extraction (SFE). Prior to purification, commercial MG oxalate purity was estimated to be about 90%. The main impurities present in SFE-purified MG oxalate were identified and quantified using HPLC–DAD. The main impurities were found to be monode-MG (monodemethylated MG oxalate synthesis impurity), 4-(dimethylamino)benzophenone (4-DMABP), MG-carbinol and LMG. The homogeneity of both reference materials was also determined. Issues associated with the stability of LMG and MG oxalate in solution forced an extensive study investigating different parameters i.e. solvent, acid, analyte concentration and temperature. MG oxalate (100 μg/mL) was found to be stable in acetonitrile containing 1% v/v glacial acetic acid for at least 155 days and LMG (100 μg/mL) was stable in acetonitrile for at least 133 days. The final purity value for MG oxalate was 94.3 ± 1.4% m/m at the 95% confidence interval (or 67% m/m if MG cation is reported). For LMG, the certified purity was found to be 98.8 ± 0.8% m/m at the 95% confidence interval. Figure Calibration reference materials for malachite green and leucomalachite green, certified for purity, are essential in characterising these key analytes in a fish matrix reference material     

Cost-effective flow cell for the determination of malachite green and leucomalachite green at a boron-doped diamond thin-film electrode.

An electrooxidation and a cost-effective flow-based analysis of malachite green (MG) and leucomalachite green (LMG) were investigated at a boron-doped diamond thin-film (BDD) electrode. Cyclic voltammetry as a function of the pH of the supporting electrolyte solution was studied. Comparison experiments were performed with a glassy carbon electrode. A well-defined cyclic voltammogram, providing the highest peak current, was obtained when using phosphate buffer at pH 2. The potential sweep-rate dependence of MG and LMG oxidation (peak currents for 1 mM MG and LMG linearly proportional to v 1/2, within the range of 0.01 to 0.3 V/s) indicates that the oxidation current is a diffusion-controlled process on the BDD surface. In addition, hydrodynamic voltammetry and amperometric detection using the BDD electrode combined with a flow injection analysis system was also studied. A homemade flow cell was used, and the results were compared with a commercial flow cell. A detection potential of 0.85 V was selected when using a commercial flow cell, at which MG and LMG exhibited the highest signal-to-background ratios. For the homemade flow cell, a detection potential of 1.1 V was chosen because MG and LMG exhibited a steady response. The flow analysis results showed linear concentration ranges of 1-100 microM and 4-80 microM for MG and LMG, respectively. The detection limit for both compounds was 50 nM.