Studies on azaspiracid biotoxins. II. Mass spectral behavior and structural elucidation of azaspiracid analogs

In this report, the mass spectral analysis of azaspiracid biotoxins is described. Specifically, the collision-induced dissociation (CID) behavior and differences between CID spectra obtained on a triple-quadrupole, a quadrupole time-of-flight, and an ion-trap mass spectrometer are addressed here. The CID spectra obtained on the triple-quadrupole mass spectrometer allowed the classification of the major product ions of the five investigated compounds (AZA 1-5) into five distinct fragment ion groups, according to the backbone cleavage positions. Although the identification of unknown azaspiracids was difficult based on CID alone, the spectra provided sufficient structural information to allow confirmation of known azaspiracids in marine samples. Furthermore, we were able to detect two new azaspiracid analogs (AZA 1b and 6) in our samples and provide a preliminary structural analysis. The proposed dissociation pathways under tandem mass spectrometry (MS/MS) conditions were confirmed by a comparison with accurate mass data from electrospray quadrupole time-of-flight MS/MS experiments. Regular sequential MS(n) analysis on an ion-trap mass spectrometer was more restricted in comparison to the triple-quadrupole mass spectrometer, because the azaspiracids underwent multiple [M + H - nH(2)O](+) (n = 1-6) losses from the precursor ion under CID. Thus, the structural information obtained from MS(n) experiments was somewhat limited. To overcome this limitation, we developed a wide-range excitation technique using a 180-u window that provided results comparable to the triple-quadrupole instrument. To demonstrate the potential of the method, we applied it to the analysis of degraded azaspiracids from mussel tissue extracts.     

Studies on azaspiracid biotoxins. I. Ultrafast high-resolution liquid chromatography/mass spectrometry separations using monolithic columns

In this study, the performance of monolithic columns was evaluated for ultrafast liquid chromatography/mass spectrometry (LC/MS) analyses and for high-resolution separations of several azaspiracid biotoxin analogs. Because of their high permeability, monolithic columns offer a number of advantages over conventional packed columns; viz., very low backpressures and relatively flat van Deemter curves at high flow rates. That is, very high flow rates can be used for ultrafast analyses or, by using longer than normal columns, high-resolution separations are possible. In a series of experiments, we varied the mobile phase flow rates between 1 and 8 mL/min, and studied their impact on chromatographic parameters such as retention time, resolution, number of plates and pressure. The chromatographic run times could be reduced to ca. 30 s without a significant change in the separation efficiency. A signal intensity comparison revealed interesting differences between atmospheric-pressure chemical ionization (APCI) and electrospray ionization (ESI) in their flow-rate dependency. An explanation with respect to the behavior as of a mass-flow or a concentration-dependent device is given in the paper. Additionally, the column length was varied between 10 and 70 cm. As a result, the number of theoretical plates increased substantially. In the example shown in the report, an increase from 13 000 plates for a 10-cm column to 80 000 for a 70-cm column is demonstrated. In addition, the potential of the monolithic columns for high-resolution LC/MS separations is shown for a complex biotoxin mixture, which was separated on a 40-cm-long column.     

Fluconazol method validation by RP-HPLC for determination in biological skin matrices

The bis-triazole antifungal fluconazole (FCZ) is used in the systemic treatment of superficial mycoses. The inconvenience of drug interactions and incidence of adverse reactions occurs in approximately 16% of patients, despite several advantages against systemic fungal infections. Because its pharmacokinetics profile is favorable to cutaneous accumulation, it presents a prominent importance in the treatment of superficial mycoses. This study shows FCZ method validation by reversed-phase high-performance liquid chromatography in the linear range of 2 to 32 microg/mL, which suitable for application in biological matrices after topical permeation studies. The method is tested in simulated FCZ alcoholic solution applied to skin extracts after in vitro permeation studies using Franz cells. Recovery shows good results (in the range 75.0% +/- 4.1% to 82.0% +/- 6.6%) regarding the biological matrices.     

Review of analytical methods for the quantification of iodine in complex matrices

Iodine is an essential element of human nutrition. Nearly a third of the global population has insufficient iodine intake and is at risk of developing Iodine Deficiency Disorders (IDD). Most countries have iodine supplementation and monitoring programs. Urinary iodide (UI) is the biomarker used for epidemiological studies; only a few methods are currently used routinely for analysis. These methods either require expensive instrumentation with qualified personnel (inductively coupled plasma-mass spectrometry, instrumental nuclear activation analysis) or oxidative sample digestion to remove potential interferences prior to analysis by a kinetic colorimetric method originally introduced by Sandell and Kolthoff ∼75 years ago. The Sandell–Kolthoff (S–K) method is based on the catalytic effect of iodide on the reaction between Ce⁴⁺ and As³⁺. No available technique fully fits the needs of developing countries; research into inexpensive reliable methods and instrumentation are needed. There have been multiple reviews of methods used for epidemiological studies and specific techniques. However, a general review of iodine determination on a wide-ranging set of complex matrices is not available. While this review is not comprehensive, we cover the principal developments since the original development of the S–K method.     

Liquid-phase microextraction for rapid AP-MALDI and quantitation of nortriptyline in biological matrices

A liquid-phase microextraction (LPME) method using a micropipette with disposable tips was demonstrated for coupling to atmospheric pressure MALDI-MS (AP-MALDI/MS) as a concentrating probe for rapid analysis and quantitative determination of nortriptyline drug from biological matrices including human urine and human plasma. This technique was named as micropipette extraction (MPE). The best optimized parameters of MPE coupled to AP-MALDI/MS experiments were extraction solvent, toluene; extraction time, 5 min; sample agitation rate, 480 rpm; sample pH, 7; salt concentration, 30%; hole size of micropipette tips, 0.61 mm (id); and matrix concentration, 1000 ppm using alpha-cyano-4-hydroxycinnamic acid (CHCA) as a matrix. Three detection modes of AP-MALDI/MS analysis including full scan, selective ion monitor (SIM), and selective reaction monitor (SRM) of MS/MS were also compared for the MPE performance. The results clearly demonstrated that the MS/MS method provides a wider linear range and lower LODs but poor RSDs than the full scan and SIM methods. The LOD values for the MPE under SIM and MS/MS modes in water, urine, and plasma were 6.26, 47.5, and 94.9 nM, respectively. The enrichment factors (EFs) of this current approach were 36.5-43.0 fold in water. In addition, compared to single drop microextraction (SDME) and LPME using a dual gauge microsyringe with a hollow fiber (LPME-HF) technique, the LODs acquired by the MPE method under MS/MS modes were comparable to those of LPME-HF and SDME but it is more convenient than both methods. The advantages of this novel method are simple, easy to use, low cost, and no contamination between experiments since disposable tips were used for the micropipettes. The MPE has the potential to be widely used in the future because it only requires a simple micropipette to perform all extraction processes. We believe that this technique can be a powerful tool for MALDI/MS analysis of biological samples and clinical applications.     

Luminescent ruthenium probe for the determination of acetyl phosphate in complex biological matrices

The first probe for the fluorogenic determination of acetyl phosphate (AcP), (bpy)(2)Ru(1,10-phenanthroline-5,6-dione dioxime) (RuPDO), was prepared and its reaction with AcP was studied in detail. The emission of the weakly luminescent RuPDO is red shifted and strongly enhanced upon reaction with AcP in the presence of metal cations like Zn(2+) or Cu(2+). The reaction occurs within 60 min incubation time under highly biocompatible conditions (aqueous buffer of pH 7, 37 °C). A linear dynamic range from 10 to 200 μmol L(-1) is observed with an LOD of AcP of 3.4 μmol L(-1) (for RuPDO-Zn). Other bio-phosphates studied show only weak interference. Furthermore, the applicability of the probe in complex biological matrices was evaluated.     

Development and validation of a rapid high-performance liquid chromatography method for the quantification of exenatide

The objective was to develop a simple HPLC method to quantify exenatide—a 39 amino acid residue incretin mimetic used in diabetes therapy. To date, only non‐validated, sometimes incomplete, gradient methods have been reported in the literature. Isocratic separation was achieved using a C₄ column and a mixed solvent system, A–B–C (48:45:7, v/v/v; pH* 5.2), where A represents KH₂PO₄ (pH 4.5; 0.1 m ) and MeCN (60:40, v/v), B corresponds to NaClO₄·H₂O (pH 6.0; 0.2 m ) and MeCN (60:40, v/v), and C is water. Exenatide eluted at 3.64 min and the total run time was 6 min. The method was specific and the response was accurate, precise and linear from 0.75 to 25 µg/mL. It was used to quantify exenatide transport across intact and laser‐porated porcine skin in vitro as a function of laser fluence [0 (i.e. intact skin), 9 and 15 J/cm², respectively]. Although no permeation was observed using intact skin, cumulative exenatide permeation after 8 h through laser porated skin was 9.6 ± 6.5 and 12.4 ± 6.4 µg/cm² at fluences of 9 and 15 J/cm², respectively. This is the first validated isocratic method for exenatide quantification and it may be of use in quality control analysis and with other biological matrices. Copyright © 2010 John Wiley & Sons, Ltd.     

Study on selective quantitative determination of barium by sulphonazo III in complex matrices

Selective quantitative determination of barium by commercially available Sulphonazo III was studied in complex matrices. The application of two more promising methods was tried, but interferences derived from cations and anions present in natural waters and waste waters made them unuseful.     

Cost effective,robust, and reliable coupled separation techniques for the identification and quantification of phospholipids in complex biological matrices: Application to insects

The quantification of phospholipid classes and the determination of their molecular structures are crucial in physiological and medical studies. This paper's target analytes are cell membrane phospholipids, which play an important role in the seasonal acclimation processes of poikilothermic organisms. We introduce a set of simple and cost‐effective analytical methods that enable efficient characterization and quantification of particular phospholipid classes and the identification and relative distribution of the individual phospholipid species. The analytical approach involves solid‐phase extraction and high‐performance thin‐layer chromatography, which facilitate the separation of particular lipid classes. The obtained fractions are further transesterified to fatty acid methyl esters and subjected to gas chromatography coupled to flame ionization detection, which enables the determination of the position of double bonds. Phospholipid species separation is achieved by high‐performance liquid chromatography with mass spectrometry, which gives information about the headgroup moiety and attached fatty acids. The total content of each phospholipids class is assessed by phosphorus determination by UV spectrophotometry. The simultaneous analysis of phosphorus, fatty acid residues, and phospholipid species provides detailed information about phospholipid composition. Evaluation of these coupled methods was achieved by application to an insect model, Pyrrhocoris apterus. High correlation was observed between fatty acid compositions as determined by gas chromatography and high‐performance liquid chromatography analysis.     

Submicromolar quantification of pyocyanin in complex biological fluids using pad-printed carbon electrodes

Pyocyanin, a toxin produced by Pseudomonas aeruginosa, offers potential as a biomarker for the indirect detection of this bacterium of major importance for infections in burns, woundcare and cystic fibrosis.Pad-printed carbon electrodes are herein explored using square wave voltammetry to detect pyocyanin in a range of buffered and biological media. Third-order polynomial baseline fitting was explored to enhance the analytical sensitivity and extend the linear range to submicromolar concentrations. These modelling baselines showed excellent correlation with the experimental data, confirmed by high Interclass Correlation Coefficients of 0.995–0.998, and enabled the quantification of pyocyanin – with linearity extended down to 0.18 μM in Human Serum and 0.336 μM in both Britton-Robinson buffer and Simulated Wound Fluid, and derived Limits of Detection of 0.17, 0.15 and 0.09 μM, respectively, in this proof-of-concept study.Therefore, the use of very simple, cost-effective printed carbon materials enabled the detection of clinically relevant concentrations of this important biomarker through a new baseline fitting model and offers a novel approach for point-of-care diagnostics where Pseudomonas aeruginosa infections are critical.     

Two-dimensional liquid chromatography/mass spectrometry set-up for structural elucidation of metabolites in complex biological matrices

For absorption, distribution, metabolism and excretion (ADME) studies of drug candidates, mass spectrometry (MS) has become an indispensable tool for the characterization of biotransformation pathways. Samples from in vivo animal studies such as plasma, tissue extracts or excreta contain vast amounts of endogenous compounds. Therefore, the generation of metabolite patterns requires dedicated sample pre-treatment and sophisticated separation methods. Methodologies used for metabolite separation are often inappropriate for structure elucidation. Therefore, a two-dimensional liquid chromatography (LC) approach in combination with MS was developed. Study samples were analyzed using high-performance liquid chromatography (HPLC) for the generation of a qualitative and quantitative metabolite pattern (first dimension) with high reproducibility and recovery without extensive sample pre-treatment. Selected radioactive metabolite fractions were then applied to micro-HPLC with off-line radioactivity monitoring and subsequent MS detection (second dimension). Applying the two-dimensional HPLC/MS approach not only major metabolites could be identified, even minor and trace metabolites were characterized. The usage of sampled metabolite fractions allowed also the re-analysis of specific metabolites for additional investigations (e.g. H/D exchange experiments or product ion scanning experiments). It could be clearly shown that the two-dimensional HPLC/MS approach showed mass spectra with higher sensitivity and selectivity significantly improving the characterization of minor and trace metabolites in in vivo ADME studies.     

Requirements for screening and confirmatory methods for the detection and quantification of marine biotoxins in end-product and official control

An overview is given of the biological origin of phycotoxins, as well as their chemical characteristics. Major poisoning types are described and examples of poisoning events are given to illustrate the importance of the phenomenon to both shellfish consumers and the shellfish producing industry. The characteristics of phycotoxins as natural products, the lack of predictability of their occurrence, economic drivers and the freshness of shellfish consumed in many countries result in a number of requirements for methods to be used in the efficient detection of these compounds. Subsequently, the performance of mouse bioassays and mass spectrometry as detection tools are compared for examples from Irish and French monitoring programmes to assess the usefulness of qualitative and quantitative tools in official control, and their fitness for purpose compared with the requirements. The final part of the paper critically reviews methods available for the end-product and official control of shellfish toxins and their use in screening and confirmatory approaches in monitoring. Recent expert consultations on the methodology for phycotoxins at European and global level are summarised and recommendations are made for future progress in this area.     

Instrumental comparison for the determination of cadmium and lead in calcium supplements and other calcium-rich matrices.

Three brands of Ca supplement, a laboratory-reagent grade CaCO3 and a certified reference material (International Atomic Energy Agency H-5 Animal Bone) wee analysed for Cd and Pb by four different analytical techniques, viz., anodic stripping voltammetry inductively coupled plasma mass spectrometry, flame atomic absorption spectrometry and electrothermal atomic absorption spectrometry. The Pb levels measured by the four techniques in the bone powder were within the certified Pb level in this certified reference material. Similarly, no significant differences [p less than 0.05; analysis of variance (ANOVA)] were observed in samples with Pb concentrations greater than 1 microgram g-1. However, the Pb levels in the laboratory-reagent grade CaCO3 obtained by flame atomic absorption spectrometry (0.79 micrograms g-1) averaged about three times higher than those measured by the other three techniques (i.e., 0.25 micrograms g-1). Although no significant differences (p less than 0.05; ANOVA) in Cd levels were observed within any of the samples (intra-sample variability), the Cd concentration measured in the different Ca supplements (inter-sample variability) varied by three orders of magnitude (ranging from 0.07 to 3.59 micrograms g-1).     

Optimization and validation of a label-free MRM method for the quantification of cytochrome P450 isoforms in biological samples

Cytochromes P450 (CYPs) play critical roles in oxidative metabolism of many endogenous and exogenous compounds. Protein expression levels of CYPs in liver provide relevant information for a better understanding of the importance of CYPs in pharmacology and toxicology. This work aimed at establishing a simple method to quantify six CYPs (CYP3A4, CYP3A5, CYP1A2, CYP2D6, CYP2C9, and CYP2J2) in various biological samples without isotopic labeling. The biological matrix was spiked with the standard peptides prior to the digestion step to realize a label-free quantification by mass spectrometry. The method was validated and applied to quantify these six isoforms in both human liver microsomes and mitochondria, but also in recombinant expression systems such as baculosomes and the HepG2 cell line. The results showed intra-assay and interassay accuracy and precision within 16 % and 5 %, respectively, at the low quality control level, and demonstrated the advantages of the method in terms of reproducibility and cost.

High-performance liquid chromatographic procedures for the determination of temafloxacin in biological matrices.

A simple and precise high-performance liquid chromatographic procedure has been developed for the determination of temafloxacin and its trace level metabolites in biological matrices. Plasma samples are ultrafiltered after addition of an internal standard in a displacing reagent containing sodium dodecyl sulfate and acetonitrile. Plasma ultrafiltrates or diluted urines are chromatographed on a reversed-phase analytical column, using an ion-pair chromatographic mobile phase and fluorescence detection. The chromatographic system allows resolution and quantitation of temafloxacin's oxidative metabolites, which collectively account for less than 2% of the administered dose. The mean intra-assay coefficient of variation for determination of temafloxacin concentrations in plasma ranging from 0.05 to 10.0 micrograms/ml was 0.7%. The procedure was implemented at four laboratories for the analysis of over 12,000 samples from clinical studies. Inter-assay coefficients of variation estimated from routine analyses of quality control samples in these studies averaged 4% or lower for concentrations in the 0.15-10 micrograms/ml range. The limit of quantitation of the procedure is approximately 10 ng/ml; inter-assay coefficients of variation at 15 ng/ml averaged under 9%. Calibration curves were reproducible and highly linear, with correlation coefficients typically averaging over 0.9995. An alternative, more complex procedure, involving methylene chloride extraction, which extends the detection limits to below 1 ng/ml, is also described.     

NMR quantification of trace components in complex matrices by band‐selective excitation with adiabatic pulses

The use of band‐selective excitation with adiabatic pulses to rapidly obtain NMR spectra of trace components in the presence of strong signals is described, along with qualitative and quantitative examples from food matrices like olive oil and honey. Copyright © 2009 John Wiley & Sons, Ltd.     

Synthesis of the ABC ring system of azaspiracid. 1. Effect of D ring truncation on bis-spirocyclization

[reaction: see text] Synthesis of a spirocyclization precursor with a truncated D ring has been accomplished. Subsequent bis-spirocyclization induced the formation of equal amounts of the natural transoidal 10R,13R bis-spirocycle and its cisoidal 10R,13S epimer under an apparent thermodynamically controlled process.