Derivatization Step Research Articles

Acrylamide Content of Turkish Black Tea, Instant and Turkish Coffee Samples

Acrylamide contents of Turkish and instant coffee and black tea samples were determined by the gas chromatography mass spectrometry without any derivatization step in this present study. The sample preparation method included two primary clean up steps. In the first step, ethanol and solutions of Carrez I and II were added into sample mixtures. The second step with solid-phase extraction C18 cartridge was carried out for clean-up. The limit of detection and limit of quantification were 1.11 and 3.66 ng/mL, respectively. The quantification level of acrylamide was determined in eight instant coffee samples in the range between 4.48 and 15.71 µg/kg while seven Turkish coffee samples had a high content of acrylamide ranging from 3.89 to 88.44 µg/kg. Acrylamide was detected in only five samples from seventeen black tea samples at a quantification level between 7.02 and 19.85 µg/kg.

Liquid chromatographic isolation of individual carbohydrates from environmental matrices for stable carbon analysis and radiocarbon dating

Carbohydrates are among the most abundant organic molecules in both aquatic and terrestrial ecosystems; however, very few studies have addressed their isotopic signature using compound-specific isotope analysis, which provides additional information on their origin (δ13C) and fate (Δ14C). In this study, semi-preparative liquid chromatography with refractive index detection (HPLC-RI) was employed to produce pure carbohydrate targets for subsequent offline δ13C and Δ14C isotopic analysis. δ13C analysis was performed by elemental analyzer-isotope ratio mass spectrometer (EA-IRMS) whereas Δ14C analysis was performed by an innovative measurement procedure based on the direct combustion of the isolated fractions using an elemental analyzer coupled to the gas source of a mini carbon dating system (AixMICADAS). In general, four successive purifications with Na+, Ca2+, Pb2+, and Ca2+ cation-exchange columns were sufficient to produce pure carbohydrates. These carbohydrates were subsequently identified using mass spectrometry by comparing their mass spectra with those of authentic standards.The applicability of the proposed method was tested on two different environmental samples comprising marine particulate organic matter (POM) and total suspended atmospheric particles (TSP). The obtained results revealed that for the marine POM sample, the δ13C values of the individual carbohydrates ranged from −18.5 to −16.8‰, except for levoglucosan and mannosan, which presented values of −27.2 and −26.2‰, respectively. For the TSP sample, the δ13C values ranged from −26.4 to −25.0‰. The galactose and glucose Δ14C values were 19 and 43‰, respectively, for the POM sample. On the other hand, the levoglucosan radiocarbon value was 33‰ for the TSP sample. These results suggest that these carbohydrates exhibit a modern age in both of these samples. Radiocarbon HPLC collection window blanks, measured after the addition of phthalic acid (14C free blank), ranged from −988 to −986‰ for the abovementioned compounds, indicating a very small background isotopic influence from the whole purification procedure. Overall, the proposed method does not require derivatization steps, produces extremely low blanks, and may be applied to different types of environmental samples.

Derivatization-free method for compound-specific isotope analysis of nonexchangeable hydrogen of 4-bromophenol.

Compound-specific isotope analysis (CSIA) is a valuable tool in environmental chemistry and in other fields of science. Currently, hydrogen CSIA of polar compounds containing exchangeable hydrogen is uncommon. To extend the scope of CSIA applications, we present an alternative method of analysis, bypassing the typical step of derivatization. The method is demonstrated for two environmental contaminants, 4-bromophenol (4BP) and 2,4,6-tribromophenol (TBP). Net isotope ratios obtained by CSIA combine the isotope composition of nonexchangeable, carbon-bound hydrogen and the exchangeable hydroxyl hydrogen. To constrain the isotope composition of the latter, an ethyl acetate solution of 4BP or TBP injected into the IRMS instrument was amended with excess water of known isotope composition. The results were calibrated using bracketing control samples analyzed in sequence with the unknown samples and the known isotope ratios of water present in ethyl acetate solution. The analytical precision was comparable to the precision for halogenated compounds without exchangeable hydrogen, analyzed using similar instrumentation. The isotope ratios of the bromophenols correlated with the isotope composition of the water in the sample matrix, suggesting that the hydroxyl group of the target compound remained close to the equilibrium with the sample water during the passage through the instrument. Based on this relationship, the signatures of the nonexchangeable hydrogen were obtained using the isotope composition of sample water as the proxy for the isotope composition of the target compound hydroxyl group. The developed method could be adopted to analysis of other low molecular weight compounds amenable to gas chromatography without the absolute need for derivatization. Currently, the method can be used for samples from laboratory experiments, with high concentrations of the target compound to provide mechanistic insight into the degradation mechanisms. Further work would be required to optimize the method to low concentration environmental samples.

LC-MS/MS measurements of urinary guanidinoacetic acid and creatine: Method optimization by deleting derivatization step

BackgroundCerebral Creatine deficiency syndromes (CCDS) include three hereditary diseases affecting the metabolism of creatine (Cr): arginine glycine amidinotransferase deficiency, guanidinoacetate methyltransferase deficiency and disorders of creatine transporter. These pathologies cause a brain creatine deficiency responsible of non-specific neurological impairments with mental retardation. LC-MS/MS measurements of guanidinoacetic acid (GAA) and creatine in urine and plasma are an important screening test to identify the deficit. Analysis of this polar and basic molecules not hold on standard column requires a derivatization step to butyl-esters. To overcome this long and fastidious derivatization, an ion pairing (IP) method was chosen in this study. MethodIP method was validated using Comité francais d'accréditation (COFRAC) recommendations. Then, urine GAA and creatine of 15 patients with a CDS deficiency suspected were tested y LC-MS/MS using IP technique, and performances were assessed with reference laboratory method (butylation method). Moreover, references values were suggested y the study of 100 urines samples of healthy patients. ResultsThe method developed provided a good accuracy and precision with intra and inter-day coefficients of variation (CVs) <15%. The curve was linear for the biological and pathological concentrations. The comparison with the reference method did not reveal any significant difference for analytical performances but showed a simplification of the preparation of samples. ConclusionThe use of IP technique that we have developed demonstrated a good correlation with the butylation method. Moreover, this new method not only allows a simplification of the technique, but also decreases in run time.

Sensitive, rapid and non-derivatized determination of glyphosate, glufosinate, bialaphos and metabolites in surface water by LC–MS/MS

Glyphosate, glufosinate and bialaphos are widely used worldwide as herbicides. It has been reported that they can transfer into environmental water bodies, posing health threat to human. Thus, a sensitive and rapid analysis methodology for these compounds and corresponding metabolites is highly necessary. Due to their zwitterionic nature, previously reported methods for determination of these compounds generally require derivatization. Also, preconcentration step is typically needed in order to obtain high sensitivity. Herein, we report a very convenient and sensitive assay for these herbicides as well as corresponding metabolites in surface water. With judicious tuning of chromatographic conditions, very low LODs were achieved without derivatization or preconcentration step. The LODs were 0.15 ng mL−1 for glyphosate, glufosinate, MPPA and bialaphos, 0.1 ng mL−1 for AMPA. The LOQs were 0.5 ng mL−1 for glyphosate, glufosinate, MPPA and bialaphos, 0.3 ng mL−1 for AMPA. Recoveries ranging in 90.3–102.8% were obtained. The intra-day relative standard deviations (RSDs) ranged in 4.0–5.6%, while the inter-day RSDs ranged in 4.7–6.7%. The ME (n = 6) ranged from 92.6 to 97.2%. This assay was applied to real samples of surface water. This method is very promising for application in determination and routine monitoring of these compounds in environmental water bodies.

A new solvent extraction method with gas chromatography–Mass spectrometry for bisphenol a determination in canned foods

A new reliable simple solvent extraction method for the endocrine disruptor bisphenol A (BPA) in canned food was developed employing an aqueous basic extraction solution of 0.25 M K2CO3/0.10 M NaOH after spiking with BPA-d16 as internal standard. The BPA was next extracted into diethyl ether after solution acidification to pH = 4 and filtration. Homogenous acetylation at dry basic conditions (acetic anhydride as derivatization agent and solvent with sodium acetate as catalyst) after diethyl ether evaporation was carried out for 30 min at 110 °C. Detection of the acetylated BPA was carried out by gas chromatography–electrospray ionization/mass spectrometry (GC–EI/MS) in the selected ion monitoring (SIM) mode with pulsed split-less mode. The method was applicable in terms of eliminating the use of solvents like acetonitrile for the extraction step, where relatively long evaporation times may have been needed to evaporate acetonitrile. Also, removing lipids and precipitating most of the proteins at acidic conditions (pH = 4) prior to diethyl ether extraction can replace the often used heptane or hexane or solid sorbents. The method was tested linear with limit of linearity (LOL = 750 μg/L) and with coefficient of determination (R2= 0.998), repeatable with relative standard deviation (RSDr &lt; 7%) with instrument detection limit (IDL) of 0.01 μg/L and limit of quantitation (LOQ) of 0.034 μg/L. The method detection limit (MDL) ranged from 0.3 μg/kg to 3.2 μg/kg based on 1 g sample (wet weight). Recovery ranged from 85% to 94% with the relative standard deviations of 2%–13%. BPA concentrations in tested canned foods from outlet stores ranged from &lt;MDL to 57.4 ± (2.6) μg/kg which were below the specific limit for BPA migration in food proposed by the European Union (EU) and within the food safety and quality criteria. The extraction and derivatization steps for BPA were unique and have not been reported in literature.

GC-QTOFMS with a low-energy electron ionization source for advancing isotopologue analysis in 13C-based metabolic flux analysis.

For the study of different levels of (intra)cellular regulation and condition-dependent insight into metabolic activities, fluxomics experiments based on stable isotope tracer experiments using 13C have become a well-established approach. The experimentally obtained non-naturally distributed 13C labeling patterns of metabolite pools can be measured by mass spectrometric detection with front-end separation and can be consequently incorporated into biochemical network models. Here, despite a tedious derivatization step, gas chromatographic separation of polar metabolites is favorable because of the wide coverage range and high isomer separation efficiency. However, the typically employedelectron ionization energy of 70 eV leads to significant fragmentation and consequently only low-abundant ions with an intact carbon backbone. Since these ions are considered a prerequisite for the analysis of the non-naturally distributed labeling patterns and further integration into modeling strategies, a softer ionization technique is needed. In the present work, a novel lowenergy electron ionization source is optimized for the analysis of primary metabolites and compared with a chemical ionization approach in terms of trueness, precision, and sensitivity.

Quantification of d-mannose in plasma: Development and validation of a reliable and accurate HPLC-MS-MS method

The present paper describes the development and the validation process – in compliance with the EMA guidelines – of a method based on tandem mass spectrometry coupled to liquid chromatography for the accurate quantification of mannose in human plasma samples. The quick sample preparation procedure, simplified by the absence of any derivatization step, makes the assay suitable for routine use in a clinical chemistry laboratory. The method validation yielded satisfactory selectivity, with a good separation of mannose from its epimers (glucose and galactose), linearity over the whole concentration range of interest (0.31–40 μg/mL), reproducibility with RSD <10%, and accuracy in the range 96–104%. Instrumental LLOD (0.31 μg/mL) and LLOQ (1.25 μg/mL) were good enough to detect endogenous plasma mannose levels and in agreement with recent data from the literature. Sensitivity was affected by a 5-fold dilution factor, which, if necessary, can be reduced. The method robustness was proven in >600 injections, most of them being of plasma samples, used also to assess the reference ranges in healthy subjects (9.93 ± 3.37 μg/mL) and type 2 diabetic patients (23.47 ± 6.19 μg/mL).

Quantitative determination of major nandrolone metabolite in human urine by gas chromatography/mass spectrometry

Nandrolone is an anabolic androgenic steroid. The use of this substance is prohibited by World Anti-Doping Agency (WADA). In National Anti-Doping Laboratory, we have developed method for quantitative determination of major nandrolone metabolite – 19-norandrosterone in human urine by GC/MS technique (Agilent 7000). Proposed method includes sample preparation of urine samples with enzymatic hydrolysis, liquid-liquid extraction followed by derivatization step with MSTFA. Deuterated 19-norandrosterone has been used as internal standard. Total run time comprised 16 min. Lower limit of quantitation accounted for 1 ng/mL. Spiked urine samples were prepared by mixing blank urine with standard solutions of 19-norandrosterone in range 1–30 ng/mL, correlation coefficient larger than 0.99. Method was verified to following validation parameters: selectivity, linearity, repeatability, accuracy, matrix effect, stability and robustness. Furthermore, measurement uncertainty was estimated. Thus, proposed method is able to detect threshold 19-norandrosterone in human urine and carry out its quantitation conforming WADA requirements.

Characterization of sugar composition in Chinese royal jelly by ion chromatography with pulsed amperometric detection

An ion chromatography coupled with pulsed amperometric detection (IC-PAD) method was developed for the determination of sugars in royal jelly (RJ). The method enables quantification of 19 sugars with higher sensitivity than HPLC method. Good linearity was observed in the range of 0.02–500 mg L–1 with coefficients of determination (R2) ≥ 0.9994. The limits of detection (LODs) and limits of quantification (LOQs) were in the range of 1.0–110 μg L–1 and 3.7–380 μg L–1, respectively. The average recoveries ranged from 91.0% to 108.0%. This simple method can be an effective alternative to GC method, which requires complicated purification and derivatization steps. It was used for the analysis of 100 Chinese RJ samples from three different areas and the results of analyzed sugars were found to be in agreement with those reported in literatures. The sugar profile results have suggested that some minor sugars are better markers for discrimination in relation to geographical origin.

Novel “turn off-on” sensors for detection of DNA-acrylamide interaction usingZnS quantum dots as a phosphorescent probe

A novel turn off-on sensor for detection of interaction between DNA and acrylamide (ACR) was developed. In this method, L-cysteine capped Mn-doped ZnS quantum dots (QDs) were used as room temperature phosphorescent probes. In the ``turn-off'' mode, ACR was absorbed onto the surface of QDs via electrostatic interaction, which caused a quenching effect of room-temperature phosphorescence signal by photoinduced electron-transfer mechanism. ACR was removed from the QDs' surface with the addition of DNA. Thus, the phosphorescence emission of QDs was recovered and the system was turned to the ``turn-on'' mode. The quenching mechanism of QDs by ACR was collisional (dynamic) and the quenching constant, binding constant, and binding site number were calculated as 3.2 x 10$^{4\, }$ M$^{-1}$, 2.04 x 10$^{4\, }$ M$^{-1}$, and 1.2, respectively. An absorption spectrometric method was also used to evaluate ACR-DNA interaction and the binding constant (K) was found as 2.4 x 10$^{5\, }$ M$^{-1}$. The developed biosensor is simple, is free of interferences coming from autofluorescence and scattering light, and does not need any derivatization step or sample pretreatment.

Quantification of Cholesterol and Cholesteryl Ester by Direct Flow Injection High-Resolution Fourier Transform Mass Spectrometry Utilizing Species-Specific Response Factors.

The quantification of free cholesterol (FC) and cholesteryl ester (CE) in mammalian samples is of great interest for basic science and clinical lipidomics. Here, we evaluated the feasibility of direct flow injection analysis (FIA) coupled to electrospray ionization high-resolution mass spectrometry (ESI-HRMS) to quantify FC and CE in lipid extracts from human serum, cultured cells, and mouse liver. Despite poor ionization efficiency of FC, the limit of quantitation was sufficient for precise and accurate quantification of FC by multiplexed HRMS (MSX) analysis without using a derivatization step. However, it was demonstrated that, upon full scan Fourier transform MS (FTMS) quantification, CE species show substantial differences in their analytical responses depending on number of double bonds, length of the acyl chain, infused lipid concentration, and other lipid components. A major determinant for these response differences is their susceptibility to in-source fragmentation. In particular, introduction of double bonds lowers the degree of in-source fragmentation. Therefore, CE species-specific response factors need to be applied for CE quantification by FTMS to achieve accurate concentrations. Method validation demonstrated that FIA-ESI-HRMS (MSX and FTMS) is applicable for quantification of FC and CE in samples used in basic science as well as clinical studies such as cultured cells, tissue homogenates, and serum.

Mapping N6 -Methyladenosine (m6 A) in RNA: Established Methods, Remaining Challenges, and Emerging Approaches.

N6 -Methyladenosine (m6 A) is the most abundant internal modification in eukaryotic mRNA. Specific m6 A reader and eraser proteins link this modification to many aspects of mRNA metabolism and regulate its levels in a dynamic way. Precise localization and quantification in varying biological samples is, therefore, relevant to understand the functional role of m6 A and mechanisms governing its regulation. In this Minireview, we summarize established and emerging concepts for m6 A mapping. Starting with the seminal m6 A-sequencing techniques based on immunoprecipitation, we will highlight technical improvements by photo-cross-linking and remaining challenges. As an alternative, antibody-free approaches will be presented. These include wild-type or engineered m6 A-sensitive enzymes and chemical biology approaches combining substrate analogues, chemical derivatization, and enzymatic steps to trace m6 A. Finally, single-molecule sequencing as a new avenue for direct detection of mRNA modifications will be discussed.

Enantioseparation of N-acetyl-dl-cysteine as o-phtaldialdehyde derivatives obtained with various primary aliphatic amine additives on polysaccharide-based chiral stationary phases

A sensitive and rapid high-performance liquid chromatography (HPLC) method was developed to enantioseparation of N-acetyl-dl-cysteine after precolumn derivatization using o-phthaldialdehyde and primary aliphatic amines. Seven polysaccharide-based chiral columns were tested in a reversed phase mode. Under the optimal chromatographic conditions, N-acetyl-dl-cysteine derivatives were completely enantioseparated on Chiralcel OZ-3R column with the resolution more than 2.5. The impact of various primary aliphatic amine additives as co-reagents (ethyl-, 1-propyl-, 1-butyl-, 1-pentylamine, (R)-sec-butylamine, tert-butylamine, isobutylamine, cyclopropyl-, cyclobutyl-, cyclopentyl and cyclohexylamine) used in precolumn derivatization step on the retention behavior (retention factor, selectivity and column efficiency) of N-acetyl-dl-cysteine derivatives was investigated. The effect of chromatographic conditions including acetonitrile content in the mobile phase, mobile phase pH, salt concentration in the mobile phase and column temperature on the retention and selectivity was investigated. The developed method was properly validated in terms of linearity, sensitivity (limit of detection and limit of quantification), accuracy, precision, intermediate precision and selectivity according to International Council for Harmonisation (ICH) of Technical Requirements for Pharmaceuticals for Human Use guidelines using internal normalization procedure. Proposed HPLC method was successfully applied to the determination of optical purity in commercially available N-acetyl-L-cysteine samples.

Potential of terracing to reduce glyphosate and AMPA surface runoff on Latosol

Glyphosate is the world’s most used herbicide and monitoring glyphosate in the environment is a relevant topic. The present study aims to develop a methodology to extract glyphosate from the soil and sediments, and assess the potential of the terracing system to mitigate contamination by glyphosate and AMPA in soil and water. Collections were performed on a weekly basis in two different periods of the agricultural calendar, totaling 24 Latosol soil samples, 12 sediment samples, and 10 water samples. The sampling was performed in two distinct areas: in the cultivation area where the lots with and without terrace were installed (soil and water of the reservoirs) and in the creek (sediment) present in the middle of the property. The analytes were extracted from the soil and sediment samples using alkaline extraction with KH2PO4 and NH4OH. The supernatant resulting from the extraction and the water samples were submitted to the derivatization (FMOC-Cl) and solid-phase extraction steps. The samples were then analyzed by high-performance liquid chromatography equipped with fluorescence detector (HPLC-FD). The soil samples showed AMPA content in all samples ranging from 0.50 to 1.11 μg g−1 of soil. Glyphosate could be quantified in 37.5% of the samples, and the concentrations ranged from 0.21 to 0.49 μg g−1. High concentrations of glyphosate were detected in the water samples (20.74 and 31.24 μg L−1) in the first rain events after application, decreasing significantly in the following rainfall events. The concentrations found were similar for both lots, but the volume of runoff water was higher in the lot without terrace, thus a greater mass of analyte was transported. None of the analytes under investigation could be quantified in the analysis of riverbed sediments. Glyphosate and AMPA, however, were detected in 50 and 75% of the sediment samples respectively. The results indicate the presence of analytes in the cultivation areas and prove the effectiveness of the terracing system in agricultural areas, limiting the dumping of the material originated from the surface runoff into water bodies and reducing the risk of contamination.

Establishment of a method for determining the origin of glutamic acid in processed food based on carbon and nitrogen stable isotope ratios

We developed a discriminant method based on the stable isotope ratio of carbon and nitrogen (δ13C and δ15N) to evaluate whether monosodium glutamate (MSG) is used in processed food samples. δ13C measurements were performed by elemental analyzer/isotope-ratio mass spectrometry (EA/IRMS) for on glutamic acid isolated from samples at high purity, and δ15N measurements were performed by gas chromatography/combustion/IRMS (GC/C/IRMS) following the purification and derivatization steps. By applying these methods, the δ13C and δ15N values for glutamic acid present in a wide variety of processed foods were obtained. Subsequently, discriminant analysis, which is a statistical analysis method, was performed by using the δ13C and δ15N values from seasoning MSG and glutamic acid from foodstuffs of known origin, and the discriminant function was derived. By substituting the measured δ13C and δ15N values of processed food samples into this discriminant function and classifying samples into two groups, seasoning MSG (the seasoning group) and glutamic acid in foodstuffs (the foodstuff group), we determined whether seasoning MSG had been used in the processed food samples. As a result, the accuracy of distinguishing between the seasoning group and the foodstuff group was very high, i.e., 96.2%, indicating that the proposed method is a highly robust and accurate method for determining whether seasoning MSG has been used in for processed foods.

Wheat Gluten Amino Acid Analysis by High-Performance Anion-Exchange Chromatography with Integrated Pulsed Amperometric Detection.

The present chapter describes an accurate and user-friendly method for determining amino acid composition of wheat gluten proteins and their gliadin and glutenin fractions. The method consists of hydrolysis of the peptide bonds in 6.0M hydrochloric acid (HCl) solution at 110°C for 24h, followed by evaporation of the acid and separation of the free amino acids by high-performance anion-exchange chromatography with integrated pulsed amperometric detection (HPAEC-IPAD). In contrast to conventional methods, the analysis requires neither pre- or post-column derivatization nor a time-consuming oxidation or derivatization step prior to hydrolysis. Correction factors account for incomplete release of Val and Ile even after hydrolysis for 24h and for losses of Ser during evaporation. Gradient conditions including an extra eluent allow multiple sequential sample analyses without risk of Glu accumulation on the anion-exchange column which otherwise would result from high Gln levels in gluten proteins.

Quantification of Underivatized Amino Acids on Dry Blood Spot, Plasma, and Urine by HPLC-ESI-MS/MS.

Enzyme deficiencies in amino acid metabolism may increase the levels of a single or several compounds in physiological fluids becoming diagnostically significant biomarkers for one or a group of metabolic disorders. Therefore, it is important to monitor a wide range of free amino acids simultaneously and to quantify them. This is time consuming if we use the classical methods and, especially now that many laboratories have introduced Newborn Screening Programs for the semiquantitative analysis, the detection and quantification of some amino acids need to be performed in a short time to reduce the rate of false positives.We have modified the stable isotope dilution HPLC-ESI-MS/MS method previously described by Qu (Qu et al., 2002) for a more rapid, robust, sensitive, and specific detection and quantification of underivatized amino acids. The modified method reduces the time of analysis to 10min with very good reproducibility of retention times and a better separation of the metabolites and their isomers.The omission of the derivatization step, enabledto achieve some important advantages: fast and simple sample preparation, exclusion of artifacts, and interferences. The use of this technique is highly sensitive and specific and allowed to monitor 40 underivatized amino acids including the key isomers and quantification of some of them, in order to cover many diagnostically important intermediates of metabolic pathways.We propose this HPLC-ESI-MS/MS method for underivatized amino acids as a support for the newborn screening as secondary test using the same dried blood spots for a more accurate and specific examination in case of suspected metabolic diseases. In this way we avoid plasma collection from the patient as it normally occurs, reducing anxiety for the parents and further costs for analysis.The same method was validated and applied also to plasma and urine samples with good reproducibility, accuracy, and precision. The fast run time, the feasibility of high sample throughput, and the small amount of sample required make this method very suitable for routine analysis in the clinical setting.

Non-conventional synthesis and photophysical studies of platinum(ii) complexes with methylene bridged 2,2'-dipyridylamine derivatives.

Methylene bridged 2,2'-dipyridylamine (dpa) derivatives and their metal complexes possess outstanding properties due to their inherent structural flexibility. Synthesis of such complexes typically involves derivatization of dpa followed by coordination on metals, and may not always be very efficient. In this work, an alternative synthetic approach, involving the derivatization step after - rather than prior to - coordination of dpa on metal center, is proposed and applied to synthesis of a number of platinum(ii) complexes with substituted benzyldi(2-pyridyl)amines. Comparison with the more conventional synthetic route reveals greater efficiency and versatility of the proposed approach. The obtained complexes are not luminescent in solution at room temperature, but display blue phosphorescence emission (ca. 415 nm) with the lifetimes of μs order in glassy matrix at 77 K, with additional green (ca. 485 nm) and relatively long living (τ = 3.7 ms) emission in the case of iodine substituted derivative.

Spectrofluorimetric determination of Bisoprolol fumarate and Rosuvastatin calcium in a novel combined formulation and in human spiked plasma

Sensitive, simple and rapid spectrofluorimetric method was developed for simultaneous determination of bisoprolol fumarate (BIS) and rosuvastatin calcium (ROS) in novel formulated tablets and in human spiked plasma depending on measuring their native fluorescence. The fluorescence intensity of BIS and ROS were measured in methanol at emission wavelength of 297 and 485 nm upon excitation at 227 and 242 nm, respectively. The emission spectrum of each drug reveals zero value at the emission wavelength of the other drug, thus allowing their simultaneous determination without any interference and without using any tedious derivatization steps. Excellent linearity was obtained over the range of 10-500 and 20-1000 ng/mL for BIS and ROS, respectively. The developed method was evaluated by applying to laboratory prepared mixtures and pharmaceutical formulation. The high sensitivity of the method was the motivation to its application for analysis of the cited drugs in spiked human plasma. Likewise, analytical and bioanalytical method validation was carried out following International Conference on Harmonisation guidelines and also statistical analysis with the reported methods was carried out and no significant difference was found. The developed method is the first developed spectrofluorimetric method for simultaneous determination of the newly formulated drugs.