Alumina Column Research Articles

Development of new cleanup method of polychlorinated dibenzo- p-dioxins/dibenzofurans in fish by freezing-lipid filtration

Freezing-lipid filtration as a new method has been developed for the rapid determination of polychlorinated dibenzo- p-dioxins/dibenzofurans (PCDDs/Fs) in biological samples. This method can effectively reduce the time of sample pretreatment, labor and amount of solvents compared with conventional methods. By freezing-lipid filtration procedure, about 90% of lipids in extract could be removed without any significant loss of PCDD/Fs. For further cleanup of extracts after freezing-lipid filtration, automatic parallel LC columns including silica gel, alumina and carbon columns were applied. During automatic parallel LC columns cleanup, most of co-extracted interferences such as residue lipids and fatty acids could be eliminated and dioxins could be separated from many other dioxin-like congeners such as polychlorinated biphenyls by this procedure. The extracts after cleanup were analyzed by high-resolution gas chromatography (HRGC)/high-resolution mass spectrometry (HRMS) using an isotope dilution method. The average recoveries and relative standard deviation (R.S.D.) of 17 native congeners in the spiked fish samples at 8–80 pg/g ( n = 3) were ranged between 85.3 and 117.2% and 5.7–20.3%, respectively.

Concentration of 99mTc-pertechnetate and 188Re-perrhenate

Summary The radioactive concentrations of 99mTc/188Re from their generators are dependent upon the specific activity of 99Mo/188W, which dictates the bed size of the alumina/gel column and the volume of physiological saline needed as eluent. Because of the high content of inactive molybdenum/tungsten in neutron irradiated MoO3/WO3, large columns containing alumina/gel are needed to produce chromatographic 99Mo → 99mTc/188W → 188Re generators. This results in large elution volumes containing relatively high 99Mo/188W contents and low concentrations of 99mTc/188Re. The decrease in radioactive concentration, or specific volume, places a limitation on some of the clinical procedures. We report post elution concentration of 99mTc/188Re using in house prepared silver cation exchange and alumina column. The high bolus volumes (10 − 60 ml saline) can conveniently be concentrated in 1 ml of saline. This approach also works very effectively to yield high specific volume solutions of 99mTc-pertechnetate from a fission 99Mo loaded alumina generator towards the end of its normal working life.

Determination of Dioxins in Atmospheric Deposition by HRGC/HRMS

A method was evaluated for the determination of seventeen 2,3,7,8-substituted polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) in atmospheric deposition with isotope dilution-high resolution gas chromatography/high resolution mass spectrometry (HRGC/HRMS). Sample extracts were cleaned up successively through multi-layer silica gel, alumina, and silica gel columns. Precision and recoveries of the entire procedure were evaluated by replicate analysis (n=4) of PCDD/Fs standard solutions, with RSD below 7.1% and the recoveries in the range of 62%-103%. The method was also validated by replicate analysis (n=3) of certified reference materials. The found values were in agreement with those certified with RSD below 15%. The detection limits were 0.1 pg for 2,3,7,8-TCDF, 0.2 pg for 2,3,7,8-TCDD, and 0.8 pg for OCDD. Using this method, PCDD/Fs were analyzed in an atmospheric deposition sample, with the deposition flux of 14.02 pg TEQ m −2·day −1.

A Simple and Practical Method for the Preparation and Purity Determination of Halide‐Free Imidazolium Ionic Liquids

AbstractThe reaction of N‐alkylimidazole with alkyl sulfonates at room temperature affords 1,3‐dialkylimidazolium alkanesulfonates as crystalline solids in high yields. The alkanesulfonate anions can be easily substituted by a series of other anions [BF4, PF6, PF3(CF2CF3)3, CF3SO3 and N(CF3SO2)2] by simple reaction of anions, salts, or acids in water at room temperature. Extraction with dichloromethane, filtration through a short basic alumina column and solvent evaporation affords the desired ionic liquids in 80–95% yield. The purity (>99.4%) of these ionic liquids can be determined by 1H NMR spectra using the intensity of the 13C satellites of the imidazolium N‐methyl group as internal standard.

Preparation of cyclotron-produced 186Re and comparison with reactor-produced 186Re and generator-produced 188Re for the labeling of bombesin

The radioisotopes 186Re and 188Re have been extensively investigated for various forms of radiotherapy due to their useful and high-abundance β particle emissions, low-abundance and imageable γ-rays, and chemical resemblance to technetium. In addition, 188Re is available in no-carrier-added (NCA) form from long lived W-188 generators, whereas 186Re can be produced in large quantities from reactors, although not in NCA form. However, NCA 186Re can be produced on a cyclotron by a ( p, n) reaction on 186W. The purpose of this study was to compare labeling of the peptide bombesin with these three forms of rhenium radioisotopes. Cyclotron-produced NCA 186Re was separated radiochemically from enriched 186W (96.9%) targets using high-purity methyl ethyl ketone (MEK). The resulting 186Re-MEK was then loaded onto a small alumina column to separate the resulting NCA 186Re from any remaining 186W. The experimental levels of impurities associated with 186Re at the end of the separation process were found to be 5.7×10 −6 Ci of 182Re (0.57%, t 1/2=12.7 h) and 1.283×10 −5 Ci of 182mRe (1.28%, t 1/2=2.67 days). The radionuclidic purity of the separated 186Re was found to be 99.6%, whereas the chemical identity was determined by reversed phase high-performance liquid chromatography (RP-HPLC) to be perrhenate ( 186ReO 4 −). Generator-produced 188ReO 4 − from a 188W/ 188Re generator (Oak Ridge National Laboratory) and CA 186ReO 4 − produced from a 185Re( n, γ) 186Re reaction at the University of Missouri Research Reactor (MURR) were used for comparison with the NCA 186Re in subsequent studies. N 3S-5-Ava-BBN(7–14)NH 2 conjugates provide flexibility for designing 186,188Re-labeled conjugates that retain high in vitro and in vivo specificity targeting of GRP receptor-expressing cells. This study showed that the N 3S-5-Ava-BBN(7–14)NH 2 could be labeled with 186,188Re following the preconjugation, postmetallation approach. The 186,188Re VO-N 3S-5-Ava-BBN(7–14)NH 2 complexes were found to form stable complexes following the reduction of perrhenate (Re VIIO 4 −) with stannous chloride at room temperature, as verified by HPLC and stability studies. The radiolabeling yield was found to be >90%. The HPLC chromatograms of 186,188Re-N 3S-5-Ava-BBN(7–14)NH 2 complexes revealed two peaks for each conjugate, reflecting the presence of syn- and anti-isomers, which were resolvable by HPLC but re-isomerized on separation. The biodistribution studies showed that the compounds were excreted through the renal and hepatobiliary systems and demonstrated receptor-specific uptake with an average pancreas accumulation of 8.15% ID/g at 1 h postinjection. Administration of cold BBN effectively blocked pancreatic uptake and further reflects the high specificity this conjugate has for the GRP receptors. At low levels of radioactivity, radiolysis effects were not observed. Scale-up may or may not elicit this effect, particularly for the higher energy β emitter 188Re. The biodistribution studies demonstrated that the CA and NCA 186,188Re conjugates behaved similarly, raising the question of whether NCA 186,188Re is necessary for specific tumor receptor targeting.

Separation of cationic aracyl derivatives of betaines and related compounds

Cationic aracyl esters of betaines can be formed by alkylation with aracyl halides or trifluoromethanesulfonates. HPLC on a non-endcapped strong cation exchange (SCX) column gave high retention of these derivatives. Cation exchange HPLC may be carried out on a normal-phase (silica or alumina) column using a polar organic solvent (acetonitrile, propan-2-ol) containing an aqueous buffer with an organic cation and a hydrophilic anion. Selectivity is affected by the choice of organic solvent and buffer, e.g. alcohols decrease the retention times of hydroxybetaines such as carnitine. Retention is reduced by increasing the water content and the buffer concentration. Capillary electrophoresis migration times are affected by the choice of buffer anion, with low pH citrate buffers favoured.

Rapid and simple determination of chloropropanols (3-MCPD and 1,3-DCP) in food products using isotope dilution GC–MS

The paper describes simultaneous determination of 3-monochloropropanediol (3-MCPD) and 1,3-dichloropropanol (1,3-DCP) in small specimens of food products. 3-MCPD and 1,3-DCP were isolated from soy sauce and other food products using alumina column extraction. Sample portions (1–2 g) spiked with deuterated 3-MCPD-d5 and 1,3-DCP-d5 as internal standards were mixed with 2 g of aluminum oxide and packed into small disposable column. The compounds were eluted with 25 ml dichloromethane, concentrated and derivatized with heptafluorobutyryl anhydride. GC–MS analysis was done in SIM mode. Four ion fragments were used for identification of 3-MCPD and 3-MCPD-d5, three ions were used for 1,3-DCP. The limit of detection was 1 ng/g, the limit of quantitation was 3 ng/g, the recovery was around 80%. Observed matrix effects were negligible for 3-MCPD and amounted to around 20% signal decrease for 3-DCP.

Evidence for numerous analogs of yessotoxin in Protoceratium reticulatum

A solid-phase extract from Protoceratium reticulatum was partitioned between water and butanol and the two fractions purified on an alumina column. Fractionation was monitored by ELISA and LC–MS. Results indicate that while almost all yessotoxin ( 1) was extracted into butanol, large amounts of yessotoxin analogs remained in the aqueous extract along with lesser amounts in the butanolic extract. NMR analysis of selected fractions from reverse-phase chromatography of the extracts confirmed the presence of yessotoxin analogs, although structure determinations were not possible due to the complexity of the mixtures. Analysis of fractions with LC–MS 3 and neutral-loss LC–MS/MS indicated the presence of more than 90 yessotoxin analogs, although structures for most of these have not yet been determined. These analogs provide a mechanism to rationalise the discrepancy between ELISA and LC–MS analyses of algae and shellfish.

Systematic analysis and overall toxicity evaluation of dioxins and hexachlorobenzene in human milk

A systematic method for analyzing dioxins (PCDDs, PCDFs and dioxin-like PCBs), hexachlorobenzene (HCB), heptachlor epoxide and β-hexachlorocyclohexane (HCH) in human milk was developed to determine the residual amount of HCB in human milk and to evaluate the overall toxicity of both dioxins and HCB in human milk. The fractionation behavior of HCB on chromatography with silica gel, alumina, and activated carbon/silica gel, and the concentrated sulfuric acid decomposition method, which is widely used as a dioxin cleanup method, were studied in order to make the preprocessing operation for HCB measurement compatible with that for conventional dioxin measurement. HCB was found to be eluted in the 2% dichloromethane (DCM)/hexane 60 ml fraction from an alumina column. Heptachlor epoxide and a part of β-HCH were eluted in the 10% DCM/hexane 50 ml fraction from a silica gel column, while the remaining β-HCH was eluted in the 25% DCM/hexane 60 ml fraction from an activated carbon/silica gel column. Moreover, HCB showed significant correlation with dioxin congeners having high toxicity equivalence factors (TEFs). The results suggest that the exposure route to HCB and its accumulation behavior in the human body are similar to those of the dioxins.

A Remarkable Ring Contraction En Route to the Chartelline Alkaloids

General procedures. All reactions were carried out under an inert nitrogen atmosphere with dry solvents under anhydrous conditions unless otherwise stated. Dry tetrahydrofuran (THF), toluene, benzene, dichloromethane (CH2Cl2), methanol (MeOH), N,N-dimethylformamide (DMF), and triethylamine (Et3N) were obtained by passing these previously degassed solvents through activated alumina columns. Reagents were purchased at the highest commercial quality and used without further purification, unless otherwise stated. Copper(I) iodide was freshly purified from refluxing saturated aqueous NaI solution (aqueous). Yields refer to chromatographically and spectroscopically (H NMR) homogeneous materials, unless otherwise stated. Reactions were monitored by thin layer chromatography (TLC) carried out on 0.25 mm E. Merck silica gel plates (60F-254) using UV light as the visualizing agent and an acidic mixture of anisaldehyde or phosphomolybdic acid or basic aqueous potassium permangante (KMnO4) and heat as developing agents. E. Merck silica gel (60, particle size 0.043–0.063 mm) was used for flash column chromatography. Preparative thin layer chromatography (PTLC) separations were carried out on 0.25 or 0.5 mm E. Merck silica gel plates (60F-254). NMR spectra were recorded on Bruker DRX-600, DRX-500, and AMX-400 or Varian Inova-400 instruments and calibrated using residual undeuterated solvent as an internal reference. The following abbreviations were used to explain the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, b = broad. High-resolution mass spectra (HRMS) were recorded on Agilent LC/MSD TOF time-of-flight mass spectrometer by electrospray ionization time of flight reflectron experiments. IR spectra were recorded on a Perkin Elmer Spectrum BX FTIR spectrometer. Melting points were recorded on a Fisher-Johns 12-144 melting point apparatus.

Receptor Activity-modifying Protein (RAMP) Isoform-specific Regulation of Adrenomedullin Receptor Trafficking by NHERF-1

Receptor activity-modifying proteins (RAMPs 1-3) are single transmembrane accessory proteins critical to various G-protein coupled receptors for plasma membrane expression and receptor phenotype. A functional receptor for the vasodilatory ligand, adrenomedullin (AM), is comprised of RAMP2 or RAMP3 and calcitonin receptor-like receptor (CRLR). It is now known that RAMP3 protein-protein interactions regulate the recycling of the AM2 receptor. The major aim of this study was to identify other interaction partners of RAMP3 and determine their role in CRLR-RAMP3 trafficking. Trafficking of G-protein-coupled receptors has been shown to be regulated by the Na+/H+ exchanger regulatory factor-1 (NHERF-1), an adaptor protein containing two tandem PSD-95/Discs-large/ZO-1 homology (PDZ) domains. In HEK 293T cells expressing the AM2 receptor, the complex undergoes agonist-induced desensitization and internalization. However, in the presence of NHERF-1, although the AM receptor (CRLR/RAMP3) undergoes desensitization, the internalization of the receptor complex is blocked. Overlay assays and mutational analysis indicated that RAMP3 and NHERF-1 interact via a PDZ type I domain on NHERF-1. The internalization of the CRLR-RAMP complex was not affected by NHERF-1 when CRLR was co-expressed with RAMP1 or RAMP2. Mutation of the ezrin/radixin/moesin (ERM) domain on NHERF-1 indicated that NHERF-1 inhibits CRLR/RAMP3 complex internalization by tethering the complex to the actin cytoskeleton. When examined in a primary culture of human proximal tubule cells endogenously expressing the CRLR-RAMP3 complex and NHERF-1, the CRLR-RAMP complex desensitizes but is unable to internalize upon agonist stimulation. Knock-down of either RAMP3 or NHERF-1 by RNA interference technology enabled agonist-induced internalization of the CRLR-RAMP complex. These results, using both endogenous and overexpressed cellular models, indicate a novel function for NHERF-1 and RAMP3 in the internalization of the AM receptor and suggest additional regulatory mechanisms for receptor trafficking.

Altered lung phospholipid metabolism in mice with targeted deletion of lysosomal-type phospholipase A2

Lung surfactant dipalmitoylphosphatidylcholine (DPPC) is endocytosed by alveolar epithelial cells and degraded by lysosomal-type phospholipase A2 (aiPLA2). This enzyme is identical to peroxiredoxin 6 (Prdx6), a bifunctional protein with PLA2 and GSH peroxidase activities. Lung phospholipid was studied in Prdx6 knockout (Prdx6-/-) mice. The normalized content of total phospholipid, phosphatidylcholine (PC), and disaturated phosphatidylcholine (DSPC) in bronchoalveolar lavage fluid, lung lamellar bodies, and lung homogenate was unchanged with age in wild-type mice but increased progressively in Prdx6-/- animals. Degradation of internalized [3H]DPPC in isolated mouse lungs after endotracheal instillation of unilamellar liposomes labeled with [3H]DPPC was significantly decreased at 2 h in Prdx6-/- mice (13.6 +/- 0.3% vs. 26.8 +/- 0.8% in the wild type), reflected by decreased dpm in the lysophosphatidylcholine and the unsaturated PC fractions. Incorporation of [14C]palmitate into DSPC at 24 h after intravenous injection was decreased by 73% in lamellar bodies and by 54% in alveolar lavage surfactant in Prdx6-/- mice, whereas incorporation of [3H]choline was decreased only slightly. Phospholipid metabolism in Prdx6-/- lungs was similar to that in wild-type lungs treated with MJ33, an inhibitor of aiPLA2 activity. These results confirm an important role for Prdx6 in lung surfactant DPPC degradation and synthesis by the reacylation pathway.

Catalytic Asymmetric Conjugate Addition of Dialkylzinc Reagents to β‐Aryl‐α,β‐unsaturated N‐2,4,6‐Triisopropylphenylsulfonylaldimines with Use of N‐Boc‐L‐Val‐Connected Amidophosphane‐Copper(I) Catalyst.

AbstractFor Abstract see ChemInform Abstract in Full Text.

HPLC method for the determination of synthetic thermal heating fluid (therminol 55TM) in basic oleochemicals

AbstractA combination of alumina column chromatography and HPLC procedures was developed for the determination of synthetic hydrocarbon‐based thermal heating fluid (trade‐marked as Therminol 55TM) in basic oleochemicals. In each run, 3 g of sample solution was loaded onto the alumina (50 g) column and was eluted with 200 mL of hexane. The eluate was then concentrated to 1 mL with the aid of a nitrogen stream prior to HPLC analysis. The HPLC chromatogram of Therminol 55 was characterized by a humped shape that could not be resolved into individual peaks. The lowest limit of quantification obtained from various spiked samples was 30 μg/g. The recovery results obtained from the method were good, ranging from 70 to 80%, whereas the overall CV were all below 10%. The method is simple and thus is ideal for quality assurance purposes.

Determination of Major Carotenoids in a Few Indian Leafy Vegetables by High-Performance Liquid Chromatography

Leafy vegetables [Basella rubra L., Peucedanum sowa Roxb., Moringa oleifera Lam., Trigonella foenum-graecum L., Spinacia oleracea L., Sesbania grandiflora (L.) Poir., and Raphanus sativus L.] that are commonly used by the rural population in India were evaluated in terms of their main carotenoid pattern. The extracted carotenoids were purified by open column chromatography (OCC) on a neutral alumina column to verify their identity by their characteristic UV-visible absorption spectra. Reverse-phase high-performance liquid chromatography (HPLC) on a C18 column with UV-visible photodiode array detection under isocratic conditions was used for quantification of isolated carotenoids. Acetonitrile/methanol/dichloromethane (60:20:20 v/v/v) containing 0.1% ammonium acetate was used as a mobile phase. The major carotenoids identified by both methods were lutein, beta-carotene, violaxanthin, neoxanthin, and zeaxanthin. Among the carotenoids identified, lutein and beta-carotene levels were found to be higher in these leafy vegetables. Results show that P. sowa and S. oleracea are rich sources of lutein (77-92 mg/100 g of dry wt) and beta-carotene (36-44 mg/100 g of dry wt) compared with other leafy vegetables. The purity of carotenoids eluted by OCC was clarified by HPLC, and they were found to be 92% +/- 3% for neoxanthin, 94% +/- 2% for violaxanthin, 97% +/-2% for lutein and zeaxanthin, and 90% +/- 3% for beta-carotene. It could be recommended to use P. sowa and S. oleracea as rich sources of lutein and beta-carotene for health benefits. The OCC method proposed is relatively simple and provides purified carotenoids for feeding trials.

Assays for Cyclic Nucleotide‐Specific Phosphodiesterases (Families 1 to 5)

AbstractThe discovery of 3′, 5′‐cyclic nucleotide phosphodiesterase (PDE) activity 40 years ago was crucial in establishing the role of cyclic nucleotides in signal transduction. At present, 11 PDE gene families have been identified, differing in substrate specificity, mechanism of regulation, tissue distribution, cellular compartmentalization, and targeting to specific signal transduction pathways. PDE enzymes are attractive drug targets for a variety of human diseases. Efficient high‐throughput PDE enzyme assays are essential for drug development, as well as for understanding the role of these enzymes in signal transduction pathways. In this unit, two types of PDE enzyme assays are described in detail. The first method is relatively inexpensive and is based on separating radiolabeled cyclic nucleotide monophosphates (cNMPs) from degradation products on alumina columns, whereas the second method is a fluorescence‐based procedure that is better suited for high‐throughput screening.

Novel Function for Receptor Activity-modifying Proteins (RAMPs) in Post-endocytic Receptor Trafficking

RAMPs (1-3) are single transmembrane accessory proteins crucial for plasma membrane expression, which also determine receptor phenotype of various G-protein-coupled receptors. For example, adrenomedullin receptors are comprised of RAMP2 or RAMP3 (AM1R and AM2R, respectively) and calcitonin receptor-like receptor (CRLR), while a CRLR heterodimer with RAMP1 yields a calcitonin gene-related peptide receptor. The major aim of this study was to determine the role of RAMPs in receptor trafficking. We hypothesized that a PDZ type I domain present in the C terminus of RAMP3, but not in RAMP1 or RAMP2, leads to protein-protein interactions that determine receptor trafficking. Employing adenylate cyclase assays, radioligand binding, and immunofluorescence microscopy, we observed that in HEK293 cells the CRLR-RAMP complex undergoes agonist-stimulated desensitization and internalization and fails to resensitize (i.e. degradation of the receptor complex). Co-expression of N-ethylmaleimide-sensitive factor (NSF) with the CRLR-RAMP3 complex, but not CRLR-RAMP1 or CRLR-RAMP2 complex, altered receptor trafficking to a recycling pathway. Mutational analysis of RAMP3, by deletion and point mutations, indicated that the PDZ motif of RAMP3 interacts with NSF to cause the change in trafficking. The role of RAMP3 and NSF in AM2R recycling was confirmed in rat mesangial cells, where RNA interference with RAMP3 and pharmacological inhibition of NSF both resulted in a lack of receptor resensitization/recycling after agonist-stimulated desensitization. These findings provide the first functional difference between the AM1R and AM2R at the level of post-endocytic receptor trafficking. These results indicate a novel function for RAMP3 in the post-endocytic sorting of the AM-R and suggest a broader regulatory role for RAMPs in receptor trafficking.

Catalytic Asymmetric Conjugate Addition of Dialkylzinc Reagents to β-Aryl-α,β-unsaturated N-2,4,6-Triisopropylphenylsulfonylaldimines with Use of N-Boc-l-Val-Connected Amidophosphane-Copper(I) Catalyst

Asymmetric conjugate addition of dialkylzinc to alpha,beta-unsaturated N-2,4,6-triisopropylphenylsulfonylaldimines 9 was catalyzed by 5 mol % N-Boc-L-valine-connected amidophosphane 5a-Cu(MeCN)(4)BF(4) in the presence of 4 angstroms MS in toluene to afford, after hydrolysis of an imine to an aldehyde through a short alumina column and reduction with sodium borohydride, the corresponding beta-alkylated alkanols 10 with 67-91% ee in reasonably high yields.

Analytical Methods for the Determination of Alkylphenolic Surfactants and Polybrominated Diphenyl Ethers in Wastewaters and Sewage Sludges. II Method Development

Analytical methods for the determination of two groups of compounds, the non-polar alkyl phenols along with their more polar ethoxylates, and the hydrophobic polybrominated flame retardants in wastewater samples have been developed. The methods allow for determination of the analytes in both the aqueous and solid phases, which were separated by centrifugation and filtration. Extraction from the aqueous phase involved the use of SPE for the surfactants, however, liquid-liquid extraction was found to be more efficient for the PBDE. Alkyl phenols and their ethoxylates were extracted from the solid phase by shaking with solvent and Soxhlet was used for extraction of PBDE. Sample cleanup was based on alumina columns for the PBDE and surfactants when extracted form solids. Quantification of surfactants utilised LC-MS with ESI in both positive and negative ionisation mode, and GC-MS (NCI) was used to determine the PBDE, with the exception of the deca congener, where a 10 m column installed on GC-ECD resulted in short run times and reduced thermal decomposition. Method recoveries for the surfactants were above 80%, and for PBDE 92±5% from the solid phase and 95±5% from the liquid phase.

Determination of malachite green residues in rainbow trout muscle with liquid chromatography and liquid chromatography coupled with tandem mass spectrometry

A method for the determination of malachite green and its major metabolite leucomalachite green in rainbow trout muscle is reported with limits of detection of 0.8 and 0.6 μg kg−1, respectively. Residues were extracted with an acetonitrile–acetate buffer mixture and partitioned into methylene chloride. Clean-up of the extracts was performed on alumina and propylsulfonic acid solid-phase extraction columns using the automated solid-phase extraction system. The chromatographic separation of malachite green and leucomalachite green was achieved on a Chromspher 5B column using an acetonitrile–acetate buffer mobile phase. Leucomalachite green was converted to malachite green by post-column oxidation before spectrophotometric detection at 600 nm. The mean recoveries of malachite green and leucomalachite green from control rainbow trout muscle spiked at 2–50 μg kg−1 were 65% (range 63.4–65.9%, relative standard deviation 3.9–16.1%) and 74% (range 58.3–82.6%, relative standard deviation 3.3–11.4%), respectively. Qualitative confirmation of the determined residues was performed with liquid chromatography coupled with tandem mass spectrometry detection with limits of detection of 2.5 and 1 μg kg−1 for malachite green and leucomalachite green, respectively.