Determination of quinolone residues in shrimp using liquid chromatography with fluorescence detection and residue confirmation by mass spectrometry

The quinolones, oxolinic acid (OXO), flumequine (FLU), and nalidixic acid (NAL), are antibacterial drugs effective against Gram-negative bacteria. Quinolones are used in both human and veterinary medicine, but are currently not approved by the U.S. Food and Drug Administration for use in food fish. A liquid chromatography-fluorescence (LC-FL) method was developed to determine OXO, FLU, and NAL residues in shrimp. An additional liquid chromatography-mass spectrometry (LC-MSⁿ) method was created to confirm these residues using the same sample extract. Samples were prepared with a simple ethyl acetate extraction followed by solvent exchange into 0.2% formic acid and cleaned-up with hexane. Reverse phase chromatography was used to separate the three compounds in both procedures. For the LC-FL determinative method, fluorescence emission was monitored at 369 nm with excitation at 327 nm. With electrospray ionization, the three most abundant ions from the MS³ product ion spectrum were used to identify OXO, FLU, and NAL in the confirmation procedure. Shrimp samples fortified at levels ranging from 7.5 to 100 ng g⁻¹ were used to validate both methods.     

Improved liquid chromatographic determination of nine currently used (fluoro)quinolones with fluorescence and mass spectrometric detection for environmental samples

An HPLC method using C18-modified silica as stationary phase has been developed for environmental trace analysis of nine (fluoro)quinolones. Detection is done by fluorescence measurement or MS using the modes of SIM and selected reaction monitoring (SRM). Best separation is achieved with a gradient consisting of 50 mM formic acid and methanol, which is fully compatible with MS coupling. LOQs (S/N of 10) for fluorescence detection are between 10 and 60 microg/L, depending on the analyte. MS detection (SIM and SRM) yields LOQs that are better by a factor of at least an order of magnitude. Sample preconcentration and sample clean-up is accomplished by SPE (preconcentration factor of 1000), leading to LOQs in the low ng/L range. Recoveries of the preconcentration procedure are better than 80% for all analytes. The suitability for real samples has been demonstrated by analyzing surface waters, municipal waste waters, sewage treatment plant effluents, sewage sludge, and sediment taken from rivers and fish ponds. The method should also be useful for determination of residues of (fluoro)quinolones in food or other matrices. The degradation of the (fluoro)quinolones has been examined over 5 days in order to get information about the decomposition rate and the degradation products eventually occurring in the environment.     

The analysis of organic mercury compounds using liquid chromatography with on-line atomic fluorescence spectrometric detection

A new method for the analysis of organic mercury compounds is reported. The organomercurials are separated by high-performance liquid chromatography (HPLC). The compounds are converted to mercury(0) in a continuous-flow system by means of an oxidizing and a subsequent reducing solution. The elemental mercury generated is swept into the cell of an atomic fluorescence spectrometer (AFS) by a stream of argon. The compositions of the oxidizing solution, which contains peroxodisulphate and copper(II) in dilute sulphuric acid, and the reducing solution, which contains alkaline tin(II) chloride, were optimized, as were the gas–liquid separator (GLS), the condensing system and the geometry of the reaction coils. The method is applied to extracts of certified reference material (CRM) and to river sediments. High concentrations of methylmercury were found in the sediment samples. At one location, the presence of ethylmercury is derived from the sample chromatogram.     

A sensitive fluorescence reagent for the determination of aldehydes from alcoholic beverage using high-performance liquid chromatography with fluorescence detection and mass spectrometric identification

A pre-column derivatization method for the sensitive determination of aldehydes using the tagging reagent 2-[2-(7H-dibenzo[a,g] carbazol-7-yl)-ethoxy] ethyl carbonylhydrazine (DBCEEC) followed by high-performance liquid chromatography with fluorescence detection and APCI-MS identification has been developed. The chromophore of fluoren-9-methoxy-carbonylhydrazine (Fmoc-hydrazine) reagent was replaced by 2-[2-(7H-dibenzo[a,g] carbazol-7-yl)-ethoxy] ethyl functional group, which resulted in a sensitive fluorescence tagging reagent DBCEEC. DBCEEC could easily and quickly labeled aldehydes. The maximum excitation (300 nm) and emission (400 nm) wavelengths did not essentially change for all the aldehyde derivatives. Derivatives were sufficiently stable to be efficiently analyzed by high-performance liquid chromatography. The derivatives showed an intense protonated molecular ion corresponding m/z [M + (CH₂)_n]⁺ in positive-ion mode (M: molecular weight of DBCEEC, n: corresponding aldehyde carbon atom numbers). The collision-induced dissociation of protonated molecular ion formed fragment ions at m/z 294.6, m/z 338.6 and m/z 356.5. Studies on derivatization demonstrated excellent derivative yields in the presence of trichloroacetic acid (TCA) catalyst. Maximal yields close to 100% were observed with a 10 to 15-fold molar reagent excess. Separation of the derivatized aldehydes had been optimized on ZORBAX Eclipse XDB-C₈ column with aqueous acetonitrile as mobile phase in conjunction with a binary gradient elution. Excellent linear responses were observed at the concentration range of 0.01-10 nmol mL⁻¹ with coefficients of >0.9991. Detection limits obtained by the analysis of a derivatized standard containing 0.01 nmol mL⁻¹ of each aldehyde, were from 0.2 to 1.78 nmol L⁻¹ (at a signal-to-noise ratio of 3).     

Comparison of liquid chromatography with fluorescence detection to liquid chromatography-mass spectrometry for amino acid analysis with derivatisation by 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate: Applications for analysis of amino acids in skin

The analysis of nineteen amino acids found in collagen was optimised using 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate (AQC) as a derivatisation reagent. The analysis and detection of nineteen AQC-amino acids using fluorescence and mass spectrometry were compared at different mobile phase pH’s and column temperatures. The pH range of the mobile phase was set between 2.7 and 6.0 and column temperatures, 15–60 °C. The majority of amino acids produced a mono-derivatised product with AQC, except cystine, lysine and hydroxylysine which were di-derivatised. Hydroxylysine’s retention time was affected most by changes in the pH, whilst hydroxyproline’s retention time was more affected by column temperature. Hydroxylysine was detected as two diastereomers which were completely resolved. The relative standard deviation of the retention times of AQC-amino acids was less than 1% and the limit of detection (LOD) and limit of quantitation (LOQ) were ranged from (0.05–0.23) µM and (0.07–0.76) µM on fluorescence and (0.02–0.10) µM and (0.06–0.33) µM on mass spectrometry respectively. This method was successfully applied for the quantitation of amino acids in different animal skins.     

Separation and characterization of oligomeric hindered amine light stabilizers using high‐performance liquid chromatography with UV and quadrupole time‐of‐flight mass spectrometric detection

Hindered amine light stabilizers are an important class of stabilizers that protect synthetic polymers from degradation and thus from changing mechanical and optical properties. The current study presents an HPLC method capable of separating oligomeric hindered amine light stabilizers on a commercially available stationary phase, employing an MS‐compatible novel mobile phase. Based on the exact masses observed with Q‐TOF‐MS, a comprehensive characterization of five different types of oligomeric hindered amine light stabilizers was achieved, leading to structural information not included in the datasheets provided by the suppliers. For the different investigated hindered amine light stabilizers, a number of recurring units up to 17 and a molecular weight of 5200 g/mol were detected. Furthermore, the analysis of stabilizer extracts of processed polypropylene samples containing different types of hindered amine light stabilizers revealed significant differences in the oligomeric pattern between standards and polymer samples. Thus, changes in the analytes’ oligomeric pattern resulting from processing or aging of polymer materials can be monitored with the presented method.     

Chromium speciation using an automated liquid handling system with inductively coupled plasma - mass spectrometric detection

The speciation of inorganic chromium in environmental samples is required for accurate assessment of pollution levels. Of the two chromium oxidation states, Cr (VI) is a known carcinogen, while Cr (III) is an essential element. Total chromium measurement cannot be used to determine actual environmental impact due to the considerable difference in toxicity of the two elemental forms. An automated liquid handling system, the PrepLab™, can be used with an inductively coupled plasma-mass spectrometer (ICP-MS) to quantify Cr (III) and Cr (VI) in liquid samples. An autosampler is used to introduce discrete sample volumes into a solid-phase chelation resin column. The Cr (III) and Cr (VI) species are separated and are introduced on-line into the VG PlasmaQuad 3 ICP-MS for detection. The chromatographic data are collected in time resolved analysis mode with the capability of simultaneous multiple-isotopic detection.     

Simultaneous qualitative assessment and quantitative analysis of flavonoids in various tissues of lotus (Nelumbo nucifera) using high performance liquid chromatography coupled with triple quad mass spectrometry

Flavonoid composition and concentration were investigated in 12 different tissues of ‘Ti-1’ lotus (Nelumbo nucifera) by high performance liquid chromatography equipped with photodiode array detection tandem electrospray ionization mass spectrometry (HPLC-DAD-ESI-MSⁿ). A total of 20 flavonoids belonging to six groups (myricetin, quercetin, kaempferol, isohamnetin, diosmetin derivatives) were separated and identified. Myricetin 3-O-galactoside, myricetin 3-O-glucuronide, isorhamnetin 3-O-glucuronide and free aglycone diometin (3′,5,7-trihydroxy-4′-methoxyflavone) were first reported in lotus. Flavonoid composition varied largely with tissue type, and diverse compounds (5–15) were found in leaf and flower stalks, flower pistils, seed coats and embryos. Flower tissues including flower petals, stamens, pistils, and, especially, reproductive tissue fruit coats had more flavonoid compounds (15–17) than leaves (12), while no flavonoids were detectable in seed kernels. The flavonoid content of seed embryos was high, 730.95 mg 100 g⁻¹ DW (dry weight). As regards the other tissues, mature leaf pulp (771.79 mg 100 g⁻¹ FW (fresh weight)) and young leaves (650.67 mg 100 g⁻¹ FW) had higher total flavonoid amount than flower stamens (359.45 mg 100 g⁻¹ FW) and flower petals (342.97 mg 100 g⁻¹ FW), while leaf stalks, flower stalks and seed coats had much less total flavonoid (less than 40 mg 100 g⁻¹ FW).     

Separation and determination of alkylglycosides by liquid chromatography with electrospray mass spectrometric detection

The separation of alkylpolyglycosides by liquid chromatography with electrospray mass spectrometric detection, using either an alkylamide or a cyanopropyl column, and acetonitrile/water mixtures as mobile phases, was developed. Using the alkylamide column and isocratic elution, the α- and β-epimers and ring isomers (pyranosides and furanosides) of the alkylmonoglycosides were resolved. The ring isomers were also resolved in a much shorter time using the cyanopropyl column with gradient elution. Using these columns, the isomers of the alkyldiglycosides and alkyltriglycosides were also partially resolved. The equilibration time was much shorter with the cyanopropyl column, which was selected to perform quantitation studies. The response factors increased more than an order of magnitude with the length of the alkyl chain, from the methyl to the decylmonoglycoside, and decrease largely for the dodecyl and tetradecylmonoglycoside. The limits of detection were of ca. 25 μM from the hexyl up to the dodecylmonoglycoside. The procedures were applied to the characterisation and determination of alkylmonoglycosides in toiletries.     

Capillary supercritical fluid chromatography with simultaneous flame ionization and mass spectrometric detection

A simple interface between a capillary supercritical fluid chromatograph and an Extranuclear Simulscan mass spectrometer is described. The SFC column is directly inserted into the ion source through the existing GC-interface. The system is equipped with a splitting device which allows simultaneous EI/MS and flame ionization detection when CO₂ is used as the supercritical phase. The effect of source temperature and pressure on CO₂ clustering was studied for optimization of source conditions. The performance of the system was evaluated with a series of model compounds and standard mixtures.     

Accelerated tryptic digestion for the analysis of biopharmaceutical monoclonal antibodies in plasma by liquid chromatography with tandem mass spectrometric detection

Accelerated tryptic digestion of a therapeutic protein including microwave irradiation and thermal transfer by convection at 60 °C and 37 °C was investigated. An analytical setup was devised to follow the protein digestion rate using 1D gel electrophoresis and liquid chromatography coupled a triple quadrupole linear ion trap mass spectrometer. The formation kinetic of its tryptic peptides was monitored in the selected monitoring mode (LC-SRM/MS). Different digestion end points (e.g. 2, 5, 10, 15, 30 and 60 min) as well as an overnight digestion were tested using a therapeutic human monoclonal antibody (mAb) with the goal of its LC-SRM/MS quantification in human plasma. The peptides from the human mAb were generated at different rates and were classified into three categories: (1) the fast forming peptides, (2) the slow forming peptides and (3) the peptides degrading over time. For many monitored peptides, a heating temperature of 37 °C with a 750 rpm mixing applied for at least 30 min provided equivalent results to microwave-assisted digestion and generally allowed the achievement of an equivalent peptide concentration as an overnight digestion carried out at 37 °C. The disappearance of the protein of the heavy and light chains can be monitored by 1D gel electrophoresis but was found not to be representative of the final tryptic peptide concentrations. For quantitative purposes a stable isotope labeled version (¹³C₄, ¹⁵N₁) of the therapeutic protein was used. The labeled protein as internal standard was found to be very efficient to compensate for incomplete digestion or losses during sample preparation.     

Analysis of anthraquinones in Rubia tinctorum L. by liquid chromatography coupled with diode-array UV and mass spectrometric detection

A liquid chromatographic (LC) method for the separation of both anthraquinone glycosides and aglycones in extracts of Rubia tinctorum was improved. For on-line MS detection atmospheric pressure chemical ionisation as well as electrospray ionisation (ESI) were used. The glycosides were ionised in both positive and negative ionisation (NI) mode, the aglycones only in the NI mode. With ESI ammonia was added to the eluent post-column to deprotonate the compounds. The efficiency of mass detection of the hydroxyanthraquinone aglycones was found to depend on the pKa value of the component. LC-diode-array detection and LC-MS provide useful complementary information for the identification of anthraquinones in plant extracts, which was proven with the identification of munjistin and pseudopurpurin.     

Direct analysis of industrial oligoglycerols by liquid chromatography with evaporative light-scattering detection and mass spectrometry

Summary A liquid chromatographic method has been developed for analysis of industrial polyglycerols, precursors of polyglycerol fatty esters, which are non-ionic surfacetants. The method utilizes two complementary chromatographic systems: porous graphitic carbon and an aminopropyl polymer with an acetonitrile-water mixture as mobile phase. Detection of these non-UV absorbing compounds was effected by means of evaporative light-scattering detection. Their structures were determined by comparison of their retention with that of synthesized standards, and by mass spectrometry.     

Ultra high performance liquid chromatography tandem mass spectrometric detection of glucuronides resistant to enzymatic hydrolysis: Implications to doping control analysis

Controversial results have been reported in the literature regarding the behavior of two testosterone (T) metabolites (3α-glucuronide-6β-hydroxyandrosterone and 3α-glucuronide-6β-hydroxyetiocholanolone) excreted after T administration. Due to their potential as biomarkers of T misuse, a UHPLC–MS/MS method for the direct quantification of these glucuronides was developed and validated. In addition, the main phase II metabolites of T that compose the steroid profile used for doping control purposes (glucuronides of T, epitestosterone, androsterone and etiocholanolone) were included. The method was found to be linear and with suitable LODs and LOQs for all metabolites. The average accuracies were between 86% and 120%, the RSDs for the intra- and inter-day precision were below 15% and 25% respectively. The method showed low matrix effect. Samples obtained before and after the administration of T were analyzed by both the developed UHPLC–MS/MS method and the GC–MS/MS method currently used by anti-doping laboratories. Relevant disagreements between the results obtained for 3α-glucuronide-6β-hydroxyandrosterone and 3α-glucuronide-6β-hydroxyetiocholanolone quantitation were observed. These markers seemed to be more suitable for the screening of T misuse when detected by UHPLC–MS/MS. These discrepancies were further investigated in 50 urine samples from healthy volunteers. The two methods gave highly correlated results for all metabolites that are currently included in the athlete's steroid profile confirming the reliability of the UHPLC–MS/MS method. However, the quantification of 3α-glucuronide-6β-hydroxyandrosterone and 3α-glucuronide-6β-hydroxyetiocholanolone, was only possible by using the UHPLC–MS/MS method since three interfering compounds were observed when performing the GC–MS/MS analysis with the most intense ion transitions. These results confirm the potential of the resistant glucuronides as biomarkers of T misuse. Additionally, they suggest that previously reported reference ranges for these metabolites should be reevaluated.     

Towards more fair and effective doping tests based on chromatography with mass spectrometric detection

Van Eenoo and Delbeke in Accred Qual Assur (2009) have criticized Faber (in Accred Qual Assur, 2009) for not taking “all factors under consideration when making his claims”. Here, it is detailed that their criticism is based on a misunderstanding of examples that were merely intended to be illustrative. Motivated by this criticism, further discussion is provided that may help in the pursuit of more fair and effective doping tests, here exemplified by chromatography with mass spectrometric detection. Surely, any doping test can only be improved or even optimized if the risks of false positives and false negatives are well defined. This requirement is consistent with a basic principle concerning mathematical approximations (Parlett in “The symmetric eigenvalue problem”, Prentice-Hall, Englewood Cliffs, 1980): apart from just being good, they should be known to be good. Author’s reply to the response on “Regulations in the field of residue and doping analysis...” Papers published in this section do not necessarily reflect the opinion of the Editors, the Editorial Board and the Publisher.     

New extraction procedure to improve the determination of quinolones in poultry muscle by liquid chromatography with ultraviolet and mass spectrometric detection

The present article aims to develop a new extraction procedure to improve the determination of quinolones in chicken muscle. This new determination method was validated using liquid chromatography–ultraviolet detection (LC–UV) and liquid chromatography–mass spectrometry detection (LC–MS), which has special bearing on stability studies. The results obtained by using the method were compared with the results obtained with a previous methodology. The new extraction procedure presents a sensitivity low enough to determine concentration of these drugs below the permissible maximum residue limits (MRL) in chicken muscle and is less time consuming than the previous methodology.     

Characterization ofMatricaria recutita L. Flower extracts by HPLC-MS and HPLC-DAD analysis

Summary Matricaria recutita L. is a spontaneous herbaceous perennial plant and its drug is largely used, as an infusion, for its anti-inflammatory properties, especially for respiratory and gastroenteric tracts. Granular soluble extracts of this drug are also used in children’s diets. The purpose of this work was to investigate the polyphenolic content of different parts of chamomile flowers. Methanolic extracts were compared and directly analysed by HPLC-DAD and HPLC-MS. The comparison between UV-Vis and MS spectra, carried out in positive and negative ionisation mode, allows identification of all the main polyphenolic compounds present in different parts of the flowers. The findings reported herein both confirm the presence of several flavonoids, described previously, and evidence large amounts of caffeic and ferulic acid derivatives. No other evidence of the presence of these compounds in chamomile flowers has been previously reported. Quantitative comparison of the flavonoid and phenolic acid derivatives present in receptacles, ligulate, tubular and total flowers was also performed.     

Assessing stability‐indicating methods for coelution by two‐dimensional liquid chromatography with mass spectrometric detection

Chromatographic analysis of trace organic impurities/degradants coeluting in the midst of active pharmaceutical ingredient can be challenging given similarities in their structures and differences in their relative levels/intensities. Conventional detection techniques such as diode array detection and mass spectrometry are often inadequate to detect/identify these residual coeluting impurities and could result in a false negative. Application of two‐dimensional chromatography to address/evaluate coelution in conventional chromatography is presented. Areas of interest, usually corresponding to the main component, are transferred to secondary column/s for further separation termed as pseudocomprehensive two‐dimensional liquid chromatography. Coelution, if any, in the rest of the chromatogram is monitored using conventional detectors. In this work, the use of similar and complementary phases in both dimensions is presented. The use of the same phase in both dimensions to resolve coeluting impurities (especially in the front and tail of the main component differing by orders of magnitude) is an easier alternative to finding complementary column/s, as hydrophobicity dominates reversed‐phase separation. The same phase separation is practical as relative levels of impurities and main component in some transferred fractions are comparable enabling their separation. The results were confirmed using mass spectrometry. This work has significant bearing as a method assessment tool in pharmaceutical and other industries.     

Isolation and determination of paspalitrem-type tremorgenic mycotoxins using liquid chromatography with diode-array detection

A liquid chromatographic (LC) method is described for the isolation and determination of the tremorgenic mycotoxins paxilline (Penicillium paxilli NRRL 6110), paspaline, paspalinine and paspalicine (Claviceps paspali). Following a Soxhlet extraction of a mould-contaminated matrix using chloroform, the crude extract was partitioned between hexane and 80% aqueous methanol. The latter fraction, containing the desired toxin(s), was evaporated to dryness, the residue dissolved in methylene chloride and the solution analysed by liquid chromatography using a Supelcosil LC-Si column eluted with methylene chloride-diethyl ether (9 + 1, v/v). A mixture containing standards of these compounds was similarly analysed. All toxins were detected using a UV diode-array detector. The generated UV spectra and chromatographic data of the standard toxins were stored in a computer as a library and used to identify these toxins in a crude mixture. The purity of the separated peaks and the amount of toxin in the crude mixture were also determined. The toxins were isolated by selectively collecting the eluted peaks using a programmable fraction collector equipped with a peak level sensor. Further confirmation of compound identity was achieved by mass spectrometry using the direct inlet probe method. In comparison with methods used previously to isolate these toxins, the present technique is fast and allows the acquisition of complete UV spectral information and chromatographic data and the isolation of multiple toxins in a single chromatographic operation.     

Development of a sensitive fluorescent derivatization reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethoxy-carbonylhydrazine and its application for determination of aldehydes from alcoholic beverage using high-performance liquid chromatography with fluorescence detection and enhance mass spectrometric identification

A pre-column derivatization method for the sensitive determination of aldehydes using the tagging reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethoxy-carbonylhydazine (BCEC) followed by high-performance liquid chromatography with fluorescence detection and enhance mass spectrometric identification has been developed. The chromophore of fluoren-9-methoxy-carbonylhydrazine (Fmoc-hydrazine) reagent was replaced by 1,2-benzo-3,4-dihydrocarbazole functional group, which resulted in a sensitive fluorescence derivatizing agent BCEC. BCEC can easily and quickly label aldehydes. The maximum excitation (333 nm) and emission (390 nm) wavelengths were essential no change for all the aldehyde derivatives. The fluorescence intensity was substantially affected by the solvents, being higher in organic than protic solvents. Derivatives are sufficiently stable to be efficiently analyzed by high-performance liquid chromatography. The derivatives showed an intense protonated molecular ion corresponding m/z [M + (CH₂)_n − 1]⁺ (M: molecular mass of BCEC, n: corresponding aldehyde carbon atom numbers) under positive-ion mode. The collision-induced dissociation of protonated molecular ion formed products at m/z = 245.7.0, m/z = 263.7 and m/z = 217.7, and corresponding the cleavage of CH₂OCO, CH₂OCO and NCH₂CH₂ bonds, respectively. Studies on derivatization demonstrated excellent derivative yields in the presence of trichloroacetic acid (TCA) catalyst. Maximal yields close to 100% were observed with a 10- to 15-fold molar reagent excess. Separation of the derivatized aldehydes has been optimized on ZORBAX Eclipse XDB-C₈ column with aqueous acetonitrile in conjunction with a binary gradient elution. Excellent linear responses were observed at the concentration range of 0.08-16.65 μmol/L with coefficients of >0.9999. Estimated detection limits for the aldehydes, obtained by successive dilution of a derivatized standard solution containing 16.65 μmol/L of each aldehyde (at a signal-to-noise ratio = 3:1), are from 3.75 to 16.65 fmol.