Validation of an UHPLC-MS/MS Method for the Determination of Malachite Green, Leucomalachite Green, Crystal Violet, and Leucocrystal Violet in Shrimp, Fish, and Salmon Muscle Using a Modified QuEChERS Approach

A quantitative and confirmatory method for detecting the presence of triphenylmethane dyes in shrimp muscle using ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) and a quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction approach was validated. The method exhibited linearity and selectivity and the coefficient of determination (R2) was higher than 0.95 for all studied analytes. Limits of detection (LODs) varied from 0.32 to 0.44 µg kg-1 and the limit of quantification (LOQ) was determined to be 0.5 µg kg-1 for all studied analytes. The trueness, precision, decision limits (CCα), detection capability (CCβ) and uncertainty presented adequate performance. In addition to the validation in shrimp muscle, fish and salmon muscle were also satisfactory validated as an extension of scope. The suitability of the proposed method was also evaluated through an interlaboratory proficiency test, in which satisfactory results were obtained. The fully validated method is thus suitable for the analysis of triphenylmethane dyes in shrimp, fish, and salmon muscle.

Dye Residue Analysis in Raw and Processed Aquaculture Products: Matrix Extension of AOAC INTERNATIONAL Official Method 2012.25

Background: Triphenylmethane dyes and metabolites are known or suspected mutagens and are prohibited in animals intended for human consumption. Despite toxicity, triphenylmethane dyes are used illegally as inexpensive treatments for fungal and parasite infections in aquatic animals. Objective: AOAC INTERNTIONAL Official Method 2012.25 for the LC-MS/MS determination of malachite green, crystal violet, brilliant green, and metabolites leucomalachite green and leucocrystal violet in seafood products was previously validated for finfish (trout, salmon, catfish, and tilapia) and shrimp, but had not been fully validated for other types of aquacultured products such as eel, molluscan shellfish, or frog or for processed seafoods. Methods: Method 2012.25 was applied to a wide scope of raw and processed aquaculture products including Arctic char, barramundi, eel, frog legs, hybrid striped bass, pompano, scallops, seabream, smoked trout, dried shrimp, and highly processed canned eel and dace products. The canned products contained oil, salt, sugar, flavorings, spices, sauces, and/or preservatives. Results: Dyes and metabolites were recovered with >85% accuracy and precision generally <20% relative standard deviation. The method detection limit was ≤0.60 μg/kg and LOQ was <1.0 μg/kg. Compounds were identified in 99% of 330 fortified and incurred samples. Conclusions: This study supports the use of Method 2012.25 for triphenylmethane dye residue analysis in a wide variety of aquacultured and seafood products. Highlights: Method 2012.25 performed well with results consistent with previous validation studies, regardless of presence of additional food ingredients or the type of processing.

Penetration Capacity, Color Alteration and Biological Response of Two In-office Bleaching Protocols

Hydrogen peroxide (H2O2) penetrates into the dental hard tissues causing color alteration but also alterations in pulpal tissues. Hard-tissue penetration, color alteration and the pulp response alterations were evaluated for two in-office bleaching protocols with H2O2. For trans-enamel/dentin penetration and color alteration, discs of bovine teeth were attached to an artificial pulp chamber and bleached according to the groups: BLU (20% H2O2 - 1x50 min, Whiteness HP Blue); MAX (35% H2O2 - 3x15 min, Whiteness HP Maxx); Control (1x50 min, placebo). Trans-enamel/dentin penetration was quantified based on the reaction of H2O2 with leucocrystal violet and the color analyzed by CIELab System. Twenty Wistar rats were divided into two groups (BLU and MAX) and their maxillary right molars were treated according to the same protocols of the in vitro study; the maxillary left molars were used as controls. After 2 days, the animals were killed and their maxillae were examined by light microscopy. The inflammation of pulp tissue was scored according to the inflammatory infiltrate (1, absent; 2, mild; 3, moderate; 4, severe/necrosis). Data were analyzed by statistical tests (α=0.05). MAX showed higher trans-enamel/dentinal penetration of H2O2 (p<0.05). The color alteration was similar for both groups (p>0.05), and different when compared to Control group (p<0.05). MAX showed severe inflammation in the upper thirds of the coronal pulp, and BLU showed moderate inflammation (p<0.05). In-office bleaching protocols using lower concentrations of hydrogen peroxide should be preferred due to their reduced trans-enamel/dentinal penetration since they cause less pulp damage and provide same bleaching efficiency.

Expansion of the Scope of AOAC First Action Method 2012.25—Single-Laboratory Validation of Triphenylmethane Dye and Leuco Metabolite Analysis in Shrimp, Tilapia, Catfish, and Salmon by LC-MS/MS

Prior to conducting a collaborative study of AOAC First Action 2012.25 LC-MS/MS analytical method for the determination of residues of three triphenylmethane dyes (malachite green, crystal violet, and brilliant green) and their metabolites (leucomalachite green and leucocrystal violet) in seafood, a single-laboratory validation of method 2012.25 was performed to expand the scope of the method to other seafood matrixes including salmon, catfish, tilapia, and shrimp. The validation included the analysis of fortified and incurred residues over multiple weeks to assess analyte stability in matrix at –80°C, a comparison of calibration methods over the range 0.25 to 4 μg/kg, study of matrix effects for analyte quantification, and qualitative identification of targeted analytes. Method accuracy ranged from 88 to 112% with 13% RSD or less for samples fortified at 0.5, 1.0, and 2.0 μg/kg. Analyte identification and determination limits were determined by procedures recommended both by the U. S. Food and Drug Administration and the European Commission. Method detection limits and decision limits ranged from 0.05 to 0.24 μg/kg and 0.08 to 0.54 μg/kg, respectively. AOAC First Action Method 2012.25 with an extracted matrix calibration curve and internal standard correction is suitable for the determination of triphenylmethane dyes and leuco metabolites in salmon, catfish, tilapia, and shrimp by LC-MS/MS at a residue determination level of 0.5 μg/kg or below.

Determination of Triphenylmethane Dyes and Their Metabolites in Salmon, Catfish, and Shrimp by LC-MS/MS Using AOAC First Action Method 2012.25: Collaborative Study

A collaborative study was conducted to evaluate the AOAC First Action 2012.25 LC-MS/MS analytical method for the determination of residues of three triphenylmethane dyes (malachite green, crystal violet, and brilliant green) and their metabolites (leucomalachite green and leucocrystal violet) in seafood. Fourteen laboratories from the United States, Canada, and the European Union member states participated in the study including national and state regulatory laboratories, university and national research laboratories, and private analytical testing laboratories. A variety of LC-MS/MS instruments were used for the analysis. Each participating laboratory received blinded test samples in duplicate of salmon, catfish, and shrimp consisting of negative control matrix; matrix fortified with residues at 0.42, 0.90, and 1.75 μg/kg; and samples of incurred matrix. The analytical results from each participating laboratory were evaluated for both quantitative residue determination and qualitative identification of targeted analytes. Results from statistical analysis showed that this method provided excellent trueness (generally ≥90% recovery) and precision (RSDr generally ≤10%, HorRat &lt;1). The Study Directors recommend Method 2012.25 for Final Action status.

Simultaneous determination of malachite green, crystal violet and their leuco-metabolites in aquaculture water samples using monolithic fiber-based solid-phase microextraction coupled with high performance liquid chromatography

Convenient, sensitive and environmentally friendly method for simultaneous monitoring of malachite green, crystal violet, leucomalachite green and leucocrystal violet in aquaculture water samples.