Current Analytical Chemistry - Volume 9, Issue 3, 2013

Lignin, the second most abundant terrestrial biopolymer, still continues to reveal fascinating complexity within its structural polymeric framework. In this regard, Pyrolysis gas chromatography mass spectrometry (Py-GC/MS) has emerged as a simple, quick and reliable analytical technique for lignin structural analysis. The literature on wide range of applications of Py-GC/MS for lignin analysis is surveyed and subsequent determination of both compositional and structural aspects of lignin polymeric framework are reviewed. The proposed mechanisms of the pyrolytic cleavage of lignin macromolecule with reference to studies of lignin model compounds are also incorporated, herein. By cleaving the complex lignin polymeric framework into characteristic smaller fragments, analytical Py-GC/MS with an extended use of gas chromatography (GC) has evolved to detect the lignin derived molecular fragments. This analytical technique offers unique leverage over other conventional and degradative methods for determining the structural features of lignin in various biological processes such as lignification and the lignin bio-degradation process. This review focuses on the recent developments in the Py-GC/MS analysis of complex lignin macromolecules and reports the characteristic structural changes of the resulting biologically modified lignin.

Stilbenes are naturally occurring plant polyphenols, which have received considerable attention due to their various biological activities, structural complexity and diversity. Rapid analysis and separation of stilbenes have been a challenge for phytochemists and analytical chemists. In recent years, major advances have been achieved by application of HPLC/MS, MS/MS and CCC. Also, many novel methods have been successfully developed. This review attempts to summarize the progress in this research area.

Phenolics, which occur widely in plants, encompass a wide range of structural classes and impart biological function to plants. They have been investigated in plants as anti-stress compounds, biosynthesized in response to abiotic and biotic stress such as pathogen attack, heavy metal, UV irradiation, and chemical stressors. In many cases, particularly for plant-metabolites interactions, analytical methods of stress driven phenolics have been intensively studied. This review focus on methodological aspects on the determination of plant defensive phenolics in response to environmental stress with a special emphasis on methods for elicitation, analysis and structural elucidation.

Marine floras contain more than 4000 polyphenolic compounds, including phlorotannins, flavonoids, anthocyanidins, lignin, tannins, catechin, epicatechin, epigallocatechin and hydroxylated polybrominated diphenyl ethers. Massive investigations have been carried out on marine polyphenols, and a great number of methods have been developed for the analyses of marine polyphenols, which possess an array of biological activities such as antioxidant, anticancer, antiinflammation, anti-diabetic, and anti-allergic. This review focuses on progress in chemical diversity, analytical methods and biological activities of marine polyphenols.

Tea is rich in polyphenol compounds, which possess antioxidant and anticarcinogenic activity, and are becoming increasingly popular because of their potential role in contributing to human health. Responding to the increasing interest in the health promoting properties of tea as well as a significant rise in scientific investigation, we have given an overview of the current analytical methods for determination of polyphenol compounds in tea samples of different origins. For this purpose, the extraction from the sample, separation and quantitative analysis of phenolic compounds involving catechins and the products of their oxidative transformations such as theaflavins and thearubigins, are covered in this review. Extraction methods evaluated include extraction at room temperature as well as heat reflux, Soxhlet, ultrasonic, supercritical carbon dioxide, microwave-assisted and high hydrostatic pressure extraction techniques. The analytical separation techniques widely employed are high-pressure liquid chromatography, gas chromatography and capillary electrophoresis. However, colorimetric or spectrophotometric methods are still the most widely used, due to their simplicity in the determination of total phenolic content, theaflavins and thearubigins, of tea.

Mentha and Thymus are important genera of the Lamiaceae family widely distributed in the entire World and commonly used in traditional medicine. Indeed, many species of the two genera have been credited with a large list of health-benefit effects, including antioxidant, anti-inflammatory, antimicrobial, analgesic, neuroprotective and anticarcinogenic. In turn, these properties have been associated to the polyphenolic composition of the plants. The present review summarizes the phenolic constituents found in Mentha and Thymus genera, as well as the main methods applied in their extraction, purification and identification. Reported species of Mentha and Thymus usually comprise derivatives of caffeic acid and distinct glycosidic forms of the flavonoids luteolin, apigenin, eriodictyol and naringenin. At present, the phenolic composition of many relevant plants of Mentha and Thymus is still unknown and thus, more studies are required for the adequate phenolic characterization of these two genera. In this context, the present implementation of faster and reliable analytical methodologies, as e.g. the chromatographic techniques hyphenated with mass spectrometry, will surely be an enormous tool in the upgrading of the missing information.

Anthocyanins are an important group of polyphenols that have health promoting properties. Analytical techniques for profiling anthocyanins have been widely reported in the last decade for in vitro and in vivo studies. A number of important technological advances in high-performance liquid chromatography (HPLC) and the introduction of the ultra high-performance liquid chromatography (UHPLC) have achieved significant gains in both the speed and the separation of anthocyanins in the past decade. Advancements in mass spectrometry, such as high-resolution and sequential collision mass spectrometry (HRMS and MSn) allow fast structural elucidation of food anthocyanins that play a very important role in food anthocyanin research. A significant increase in the number of studies has been reported for tandem MS, HRMS, and MSn based approaches for food analysis. This article provides an updated review of the application of liquid chromatography (HPLC and UHPLC) in combination with mass spectrometry (MS) for the study of food anthocyanins in recent years.

Phenolics are related to a number of pharmacological effects and form an important category among secondary plant metabolites. For many years well established and highly efficient methods such as UV spectroscopy, high performance liquid chromatography, gas chromatography, liquid chromatography, mass spectrometry and capillary electrophoresis have been used for determination. In the last years, near infrared spectroscopy with its benefits such as being noninvasive, rapid, almost no necessary sample preparation, on-/inline measurements, being able to determine physical and chemical parameters simultaneously, became a widely used analytical technique in phyto-analytics and is included in pharmacopeias to an increasing extent [1]. This manuscript reviews recent applications of NIRS in the analysis of phenolic compounds in plant material.

Polyphenols, good antioxidants, exist widely in plants and food resources. The separation methods of polyphenols from crude food and plant materials are reviewed. The separation methods reviewed include the pre-concentration by extraction methods (traditional extraction, SPE, and other extraction methods) and further separation by chromatography (HPLC, UHPLC, GC, column chromatography, TLC, and CCC) or other methods (like CE). Different materials, solvent systems, and separation conditions are discussed in this review. Publications since 2000 are included.

Styrene is a commercially important chemical widely used, whose metabolic pathway in humans leads to mandelic (MA) and phenylglyoxylic (PGA) acids, whose total concentration is the dose biomarker suggested by ACGIH for occupational exposure. Non occupational exposure to styrene is uncommon, but nevertheless a background value is always found in human urine. Most analytical methods applied to workers biomonitoring use HPLC-UV, a non specific analytical technique. The reference value reported for non styrene exposed Italian population was determined by HPLCMS/ MS but without considering matrix effect, an alteration of ionization efficiency due to the presence of undetected coeluting substances that can affect the reliability of results. This work presents the validation of quantitative determination by HPLC-MS/MS for MA and PGA using the isotopic dilution method that makes possible the determination of urinary concentration compensating for the matrix effect, and the comparison of this results with those obtained with a traditional HPLC/UV method. LOD is 0.02 mg/l for MA and 0.015 mg/l for PGA, and LOQ 0.075 and 0.040 mg/l respectively. Accuracy is always higher than 82% and variability lower than 11% for both analytes, while a very strong matrix effect makes the use of the internal standard crucial, in order to achieve reliable results. Using this method, we showed that HPLC/UV method overestimates the biomarkers levels because of its poor specificity, while MS/MS detection presents the risk of underestimation because of the urinary matrix, if this is not correctly compensated.

The aims of this research were to develop and validate a simple, sensitive method to determine un-conjugated urinary cotinine (COT-U) levels and to investigate its ability to discriminate active and environmental tobacco smoke (ETS) exposure. For this assay, urine was diluted with water, cotinine-d3 was added as an internal standard, and the sample was separated by a C18 column and analysed by triple quadrupole mass spectrometry. The quantification limit was 0.1 μg/L, range of linearity was 0.1–4000 μg/L, intra- and inter-run precision were <10%, and accuracy was within 13% of the theoretical value. Investigation of the matrix effect showed that the internal standard controlled sources of bias. The assay was applied to 168 adults who classified themselves as non-smokers with (9.5%) or without (67.9%) ETS exposure, active smokers (20.2%), and those who did not report smoking information (2.4%). Median COT-U levels were 1.3, 0.6, 687, and 57 μg/L, respectively. Based on a critical evaluation of self-classification and COT-U levels, we proposed a 30- μg/L cut-off value to identify active smoking. The ability of COT-U levels to distinguish ETS exposure was evaluated among non-smokers, but a wide overlap between groups with and without ETS exposure prevented the identification of a reliable cut-off value. A 2-μg/L COT-U cut-off value correctly identified 95.4% of self-classified non-ETS exposure and 33.3% of self-classified ETS exposure. This method reliably measured a wide range of COT-U levels. The 30-μg/L cutoff value appropriately classified active tobacco smoke exposure, but the classification of ETS exposure needs further research.

The thiobarbituric acid (TBA) reaction with malondialdehyde (MDA) is commonly used to measure free radical- mediated oxidative changes in lipids containing polyunsaturated fatty acids (PUFA). This test has had a broad range of applications since it was introduced in 1949; among others, researchers have used this test to evaluate toxic outcomes of chemical exposure in many environmental and occupational settings. However, the test has been criticized because of its low specificity. It has been reported that several endogenous or exogenous compounds, including drugs and metals, may interfere with a photometric or fluorimetric assay. The wide use of this test in occupational toxicology to measure lipid peroxidation (LPO) in humans led us to investigate whether simultaneous medications with drugs or exposure to metals interfere with the assay. We show here that, in the presence of bismuth or medazepam, the test gives erroneous results in a spectrophotometric assay. Among the tested 45 compounds, bismuth inhibits the formation of the MDA-TBA complex. In contrast, medazepam caused 2-fold higher absorbance compared to the absorbance obtained in the absence of the drug. UV spectrum analysis revealed that another unknown product is formed with an absorption maximum of 458 nm. In addition, medazepam yielded a second product with TBA whose absorption spectrum was essentially identical to that of the MDA standard. We have succeeded in separating this product and quantifying the MDA-TBA complex accurately by a HPLC method. Though it requires more sophisticated equipment, we suggest using HPLC for MDA quantification in biological samples, which might contain potentially interfering compounds.

Biological monitoring usually relies on the collection of blood and urine samples. Although being non-invasive and providing an inextinguishable sampling pool, the analysis of exhaled breath is not well established. A gas phase measurement is, however, inherently simpler than the analysis of complex biological fluids, and modern methods have identified hundreds of volatile compounds in the breath of persons exposed to normal environmental concentrations. The most commonly deployed analytical techniques in breath analysis are gas chromatography combined with mass spectrometry (GC/MS) and other MS-based methods. Lately, also laser-based optical methods, such as cavity ring-down spectroscopy (CRDS), have emerged in the field. With such instruments, it is possible to accurately quantify the concentrations of volatiles in exhaled breath down to below part-per-billion (ppb) levels with sub-second time resolution. Laser spectroscopy thereby enables real-time investigations during and after exposure to exogenous chemicals. In general, depending on the sampling approach used, the measured levels of the breath compounds may vary significantly. It is therefore of importance to systematically study and account for the phenomena affecting the recorded concentrations, and subsequently select an appropriate sampling and measurement strategy. In Helsinki, we have used CRDS to study the background levels of hydrogen cyanide (HCN), ammonia (NH3) and acetylene (C2H2) in the exhaled breath of healthy volunteers. Different sampling techniques have been employed in an effort to standardize the breath sampling event. The realtime elimination kinetics of breath C2H2 after smoking has also been studied.

Formaldehyde (FA) ranks 25th in the overall U.S. chemical production, with more than 5 million tons produced each year. Given its economic importance and widespread use, many people are exposed to FA occupationally. Recently, based on the correlation with nasopharyngeal cancer in humans, the International Agency for Research on Cancer (IARC) confirmed the classification of FA as a Group I substance. Considering the epidemiological evidence of a potential association with leukemia, the IARC has concluded that FA can cause this lymphoproliferative disorder. Our group has developed a method to assess the exposure and genotoxicity effects of FA in two different occupational settings, namely FAbased resins production and pathology and anatomy laboratories. For exposure assessment we applied simultaneously two different techniques of air monitoring: NIOSH Method 2541 and Photo Ionization Detection Equipment with simultaneously video recording. Genotoxicity effects were measured by cytokinesis-blocked micronucleus assay in peripheral blood lymphocytes and by micronucleus test in exfoliated oral cavity epithelial cells, both considered target cells. The two exposure assessment techniques show that in the two occupational settings peak exposures are still occurring. There was a statistical significant increase in the micronucleus mean of epithelial cells and peripheral lymphocytes of exposed individuals compared with controls. In conclusion, the exposure and genotoxicity effects assessment methodologies developed by us allowed to determine that these two occupational settings promote exposure to high peak FA concentrations and an increase in the micronucleus mean of exposed workers. Moreover, the developed techniques showed promising results and could be used to confirm and extend the results obtained by the analytical techniques currently available.

New separations methods are required that meet requirements of contemporary technology such as low cost, speed, and portability of instruments. Ion mobility spectrometry (IMS) complies with these requirements. In this work, IMS was modified by introducing nitrobenzene (NB) into the buffer gas of a mobility spectrometer to differentially increase the drift times of test compounds. Ion mobilities of tetramethylammonium (TMA), tetraethylammonium (TEA), tetrapropylammonium (TPA), and tetrabutylammonium (TBA) ions, 2,4-dimethyl pyridine (2,4-lutidine), 2,6-di-tert-butyl pyridine (DTBP), ethanolamine, atenolol, and valinol decreased depending on their structures. We used an ion mobility spectrometer with electrospray ionization coupled to a quadrupole mass spectrometry. The drift times of the analytes increased according to the amount of NB introduced into the buffer gas and analyte structure. When the amount of NB increased from 0.0 to 1.0 mmol m-3, percent increases in drift times were: ∼ 80% (water clusters), 62% (ethanolamine), 24% (valinol), 15% (2,4-lutidine), 2.6% (arginine), 2.1% (atenolol), 0.9% (TMA), 0.4% (TPA), and 0.2% (DTBP, TEA, and TBA). These differences in drift time change were due to formation of large analyte ion-NB clusters. The small change in mobility of tetraalkylammonium ions and DTBP with the introduction of NB into the buffer gas was explained by steric hindrance of bulky substituents which shielded the positive charge of the ion from the attachment of NB molecules, and delocalized the positive charge. NB in the buffer gas produced ion clusters with one or two NB molecules in compounds with little steric hindrance such as ethanolamine.

Bee losses associated with colony collapse disorder (CCD) has become a serious worldwide problem. Over 60 contributing factors of CCD have been identified, being one of them the pesticide exposure, and particularly to neonicotinoid pesticides as imidacloprid. So, it is interesting to study its possible presence in beeswax, as this product could be a storage location of this pesticide not only for its original beehive, but for other beehives where some of the beeswax could be used as foundation. A simple and fast method has been developed to measure imidacloprid in beeswax based on liquid chromatography (LC) coupled to an electrospray ionization mass spectrometry (ESI-MS) detection. Beeswax was diluted in a nhexane/ isopropanol mixture, and it was performed a liquid-liquid extraction with water. The aqueous phase was passed through a diatomaceous material based cartridge, and the analytes were eluted with acetone. This eluate was evaporated to dryness and reconstituted with a mixture of 0.1% formic acid and acetonitrile. A Kinetex C18 analytical column (150 × 4.6 mm i.d.) was used and the mobile phase selected was a 50:50 (v/v) mixture of water at pH 7 and methanol in isocratic elution mode. This method was fully validated in terms of selectivity, linearity, precision and trueness and the limits of detection (LOD) and quantification (LOQ) were 0.1μg/kg and 0.5 μg/kg respectively. Finally, the proposed method has been applied to the analysis of beeswax samples from Spanish apiaries located close to fruit trees.

This study represents the first report on the development and validation of a highly sensitive fluorimetric method for determination of cinacalcet hydrochloride (CIN) in tablets and plasma. The method was based on nucleophilic substitution reaction of CIN with 7-chloro-4-nitrobenzoxadiazole (NBD-Cl) in an alkaline buffered medium (pH 9) to form a highly fluorescent derivative that exhibited maximum fluorescence intensity at 552 nm after excitation at 469 nm. The factors affecting the reaction were carefully optimized. The kinetics of the reaction was investigated, the stoichiometry of the reaction was determined, and the mechanism was postulated. The activation energy of the reaction was determined and found to be 38.77 KJ/mole. Under the optimum reaction conditions, a linear relationship with good correlation coefficient (r = 0.9999) was found between the fluorescence intensity and CIN concentrations in the range of 5—200 pg/mL. The limits of detection and quantitation were 2.2 and 6.6 pg/mL, respectively. The method was reproducible as the relative standard deviations of the results did not exceed 2%. The proposed method was successfully applied to the determination of CIN in its tablets with good accuracy; the label claim percentages were 102.81–103.39 ± 1.43–3.23% (RSD). The high sensitivity of the method allowed its successful application to the accurate determination of CIN in spiked human plasma without extraction procedures; the recovery was 102.93–104.07 ± 0.04–0.53% (RSD). The proposed method is valuable for routine application in quality control and clinical laboratories for determination of CIN.

The adsorption parameters of Pb(II) on the Amberlyst 36 were investigated and solid phase extraction was applied for the preconcentration of lead ions. Flame atomic absorption spectrometry was used for detection. The recovery of lead at optimum parameters such as pH (2), elution solution type and volume (5 mL of 3 mol/L HCl solution), flow rate (3 mL/min) etc. was obtained as (98.4±1.6) % at confidence level of 95% for eight measurements. The relative standard deviation of the recovery was about 1.9%. The analytical detection limit calculated by dividing the instrumental detection limit by the enrichment factor (150) was 0.68 μg/L. The matrix effect on the adsorption was also investigated and the application of proposed method to the real samples was done. The accuracy of the method was controlled with applying the standard reference material (CWW-TM-D Waste Water) containing lead. Kinetic studies were performed to determine the kinetic parameters and the thermodynamic parameters. The process of lead adsorption was found to be follow pseudo second- order rate expression and obey the Langmuir's isotherm model with the maximum adsorption capacity of 232.6 mg/g at 328 K. Thermodynamic parameters such as the changes of standard free energy (ΔG°), enthalpy (ΔH°) and entropy (ΔS°) were also evaluated. According to results, the adsorption of Pb(II) on to Amberlyst 36 is a spontaneous, endothermic and ion-exchange process.