Conventional Spectrophotometric Method Research Articles

Application of Multivariate Calibration Methods, in Dissolution Testing and Simultaneous Determination of Atorvastatin and Ezetimibe in Their Combined Solid Dosage Form

Background: Two simple, precise and accurate multivariate calibration methods, partial least square (PLS) and principal component regression (PCR) have been applied for the simultaneous determination and dissolution profile evaluation of atorvastatin (ATV) and ezetimibe (EZT) in their binary mixtures and commercial tablets. Due to the closely overlapping spectral bands of the mentioned drugs, simultaneous determination without previous separation is not possible by conventional spectrophotometric methods. In the proposed methods (PLS and PCR) determination of chemicals was performed by the use of a full-spectrum multivariate calibration method. Methods: The experimental calibration matrix was designed orthogonally with 16 samples composed of different mixtures of both compounds in related mediums. The simultaneous determination of ATV and EZT was accomplished in mixtures through recording the absorption spectra within a range of 210 to 300 nm. Results: The concentration of ATV and EZT were considered in the linear range, between 8 to 14 µg.ml-1. The specificity of the methods was evaluated by analyzing laboratory prepared mixtures of the mentioned drugs in specific proportions. Conclusion: The applied methods were successfully employed in simultaneous spectrophotometric determination and dissolution profile evaluation of ATV and EZT in their prepared mixtures and pharmaceutical formulation.

Flow Injection Determination of Lactate Using Immobilized Lactate Dehydrogenase Enzyme with Tris(2,2′-Bipyridyl)Ruthenium(III) Chemiluminescence Detection

ABSTRACTA flow-injection procedure is reported for the determination of L-lactate based on its enhancement on tris(2,2′-bipyridyl)ruthenium(III) chemiluminescence in the presence of reduced nicotinamide adenine dinucleotide produced using immobilized lactate dehydrogenase. The calibration graph was linear between 4 and 50 × 10−7 mol L−1 (r2 = 0.9998) with relative standard deviation from 1.6 to 3.0% (n = 4). The limit of detection was 1 × 10−7 mol L−1 with a sample throughput of 60 per hour. The effect of common organic compounds and metal ions was studied. The immobilized lactate dehydrogenase exhibited good operational activity at 37°C for one week and storage stability at 4°C over a period of 10 weeks. The method was applied to blood serum, and the results were in good agreement with those obtained by a conventional spectrophotometric method with no significant difference at the 95% confidence level.

Determination of Palladium in Water Samples Using Cloud Point Extraction Coupled with Laser Thermal Lens Spectrometry

A preconcentration procedure for determination of palladium by laser thermal lens spectrometry (TLS) is proposed. It is based on cloud point extraction of palladium(II) ions as 2-(3,5-dichloro-2-pyridylazo)-5-dimethylaminoaniline (3,5-diCl-PADMA) complexes using octylphenoxypolyethoxyethanol (Triton X-114) as surfactant. The effects of various experimental conditions such as pH, concentration of ligand and surfactant, equilibration temperature and time on cloud point extraction were studied. Under the optimized conditions, the calibration graph was linear in the range of 0.15~6 ng mL^−1, and the detection limit was 0.04 ng mL^−1 with an enrichment factor of 22. The sensitivity was enhanced by 846 times when compared with the conventional spectrophotometric method. The recovery of palladium was in the range of 96.6%~104.0%. The proposed method was applied to the determination of palladium in water samples.

Variability, toxicity, and antioxidant activity of Eupatorium cannabinum (hemp agrimony) essential oils

Content: Eupatorium cannabinum L. (Asteraceae) is as a potential source of biologically active compounds. The plant is used in traditional medicine for the treatment of diarrhea and livers diseases.Objective: The present study provides investigation on pharmacological properties (antioxidant and toxic activities) of essential oils of E. cannabinum, collected from 11 wild populations in Lithuania.Materials and methods: Twenty-two hemp agrimony essential oil samples were prepared by hydrodistillation according to the European Pharmacopoeia, and their chemical composition was determined by GC–FID and GC–MS. Compositional data were subjected to principal components analysis (PCA). Instead of conventional spectrophotometric methods, cyclic voltammetry (CV) and square wave voltammetry (SWV) techniques were applied to determine antioxidant activity of hemp agrimony essential oils. Meanwhile, toxicity of the oils was determined using brine shrimp (Artemia sp.) assay.Results: Chemical profiles of E. cannabinum oils were described according to the first predominant components: germacrene D (≤22.0%), neryl acetate (≤20.0%), spathulenol (≤27.2%), and α-terpinene (11.5%). For the first time, α-zingiberene (≤7.8%) was found to be among three major constituents (as the second one) for hemp agrimony oils. SWV measurements revealed that oxidation potentials of compounds present in the oils are lower (below 0.1 V) compared with that of well-known antioxidant quercetin (0.15 V). Toxicity tests evaluated that hemp agrimony oils containing predominant amounts of germacrene D and neryl acetate were notably toxic (LC50 value 16.3–22.0 μg/mL).Conclusion: The study provided some new data concerning chemical composition and pharmaceutical properties of E. cannabinum essential oils.

A comparative study of the novel spectrophotometric methods versus conventional ones for the simultaneous determination of Esomeprazole magnesium trihydrate and Naproxen in their binary mixture

Two novel simple, specific, accurate and precise spectrophotometric methods manipulating ratio spectra are developed and validated for simultaneous determination of Esomeprazole magnesium trihydrate (ESO) and Naproxen (NAP) namely; absorbance subtraction and ratio difference. The results were compared to that of the conventional spectrophotometric methods namely; dual wavelength and isoabsorptive point coupled with first derivative of ratio spectra and derivative ratio. The suggested methods were validated in compliance with the ICH guidelines and were successfully applied for determination of ESO and NAP in their laboratory prepared mixtures and pharmaceutical preparation. No preliminary separation steps are required for the proposed spectrophotometeric procedures. The statistical comparison showed that there is no significant difference between the proposed methods and the reported method with respect to both accuracy and precision.

Development of multivariate calibration method for simultaneous determination of nickel, lead and zinc in tap water

Conventional spectrophotometric methods for simultaneous determination of nickel, lead and zinc in forms of complexes with a reagent is not feasible due to the overlap of their absorption spectra. A multivariate calibration method was used to overcome this problem. In this study, the calibration model was constructed based on absorption spectra of 30 mixture standards in the range from 490 to 600 nm. Factors influencing experimental results such as amount of reagents, pH, and color development time were optimized. The standard calibration ranges for determination of nickel, lead and zinc were found at 0.5-5 ppm. The method was applied for determination of these ions in tap water samples at ppm level, with recoveries (and RSD) of nickel, lead and zinc were 103.3 % (3.0 %), 74.9 % (11.5 %) and 104.6 % (4.6 %), respectively.

Novel Method for the Analysis of Ribonuclease Based on Fluorescence Recovery of a Cationic Aluminum Phthalocyanine-RNA Association Complex as a Red-emitting Fluorogenic Substrate.

The conventional spectrophotometric method that is often applied to determine ribonuclease (RNase) has disadvantages that include cumbersome manipulation, time-consuming processing and a lack of linear range. We had found that a low concentration of RNA could induce cationic aluminum phthalocyanine (tetra(trimethylammonio)aluminum phthalocyanine (TTMAAlPc)), which emitted strong red fluorescence to aggregate in neutral media, resulting in an almost complete quenching of fluorescence from the cationic aluminum phthalocyanine. The RNA is degraded through hydrolysis by RNase, which destroys the induced aggregation of TTMAAlPc on RNA and releases free TTMAAlPc, leading to a significant fluorescence recovery of the reaction system. Based on this new finding, a method to detect RNase by enhanced fluorescence was established using the TTMAAlPc-RNA association complex as a new fluorogenic substrate of RNase. The optimal conditions were determined, and the interfering foreign substances were investigated. Under optimal conditions, the linear range was 0.05 - 50 μg/L, and the detection limit was 0.02 μg/L. This method was applied for the analysis of ribonuclease in urine specimens from normal adults, and the results were consistent with those determined by conventional spectrophotometric methods. The developed method is easy to operate and highly sensitive, and has a wide linear range, thus solving issues with conventional methods. This study applied, for the first time, cationic phthalocyanine as a fluorescent probe in the detection of nuclease, which provides new applications of phthalocyanine as a fluorescent probe emitting at the red wavelength region.

High-Throughput Screening for a Moderately Halophilic Phenol-Degrading Strain and Its Salt Tolerance Response

A high-throughput screening system for moderately halophilic phenol-degrading bacteria from various habitats was developed to replace the conventional strain screening owing to its high efficiency. Bacterial enrichments were cultivated in 48 deep well microplates instead of shake flasks or tubes. Measurement of phenol concentrations was performed in 96-well microplates instead of using the conventional spectrophotometric method or high-performance liquid chromatography (HPLC). The high-throughput screening system was used to cultivate forty-three bacterial enrichments and gained a halophilic bacterial community E3 with the best phenol-degrading capability. Halomonas sp. strain 4-5 was isolated from the E3 community. Strain 4-5 was able to degrade more than 94% of the phenol (500 mg·L−1 starting concentration) over a range of 3%–10% NaCl. Additionally, the strain accumulated the compatible solute, ectoine, with increasing salt concentrations. PCR detection of the functional genes suggested that the largest subunit of multicomponent phenol hydroxylase (LmPH) and catechol 1,2-dioxygenase (C12O) were active in the phenol degradation process.

Development of an analytical method to determine malondialdehyde as an oxidative marker in cryopreserved bovine semen

A new methodology more sensitive to determine MDA in cryopreserved bovine semen as a biomarker of oxidative stress was developed and validated by HPLC in comparison to the conventional spectrophotometric method.

Metal-mediated protein oxidation: applications of a modified ELISA-based carbonyl detection assay for complex proteins.

Therapeutic proteins are prone to oxidative modification during manufacturing, processing, and storage that may lead to degradation, aggregation, and immunogenicity. Protein carbonylation is an irreversible oxidative modification and has been identified as a hallmark of severe oxidative stress but not extensively studied for its impact on the stability and activity of therapeutic proteins. We describe the application of a modified ELISA-based method to quantify global levels of carbonyl modification of complex proteins. We investigated protein oxidation of large protein molecules (transferrin, rabbit IgG, or β-glucosidase) and complex protein samples (human plasma) that were either stored in different buffer formulations, with varying amounts of divalent iron, or under different storage temperatures to determine the impact of different physicochemical stresses on carbonyl modifications. The modified ELISA allows for sensitive and specific carbonyl quantification with measurements that closely match those determined with the conventional spectrophotometric method. The method was useful for complex protein mixtures such as cell lysates without the need for additional procedures to remove DNA and RNA. Our findings demonstrate significant oxidative modification of each of the proteins stored in commonly used buffers and excipients at 37°C, 23°C, and 4°C. The carbonyl levels were further exacerbated with addition of trace amounts of Fe(2+). We also measured the extent of protein aggregation under oxidizing conditions. Collectively, our results indicate the importance of better characterizing carbonyl modification of proteins during their storage and use.

Charge transfer complexes beneficial method for determination of fluoxetine HCl pure drug: Spectroscopic and thermal analyses discussions

A simple and conventional spectrophotometric method is developed for quantitative analysis of fluoxetine. The method is based on the charge transfer 1: 1 complex formation of fluoxetine hydrochloride with electron acceptors: picric acid, dinitrobenzene, p-nitrobenzoic acid, 2,6-dichloroquinone-4-chloroimide, 2,6-dibromoquinone-4-chloroimide and 7,7′,8,8′-tetracyanoquinodimethane. The charge-transfer complexes are isolated and characterized by elemental analysis, conductivity, IR, Raman, 1H NMR spectra, X-ray powder diffraction, scanning electron microscopy and thermogravimetric analysis. The formation constants (KCT), molar extinction coefficients (ɛCT), standard free energies (ΔG0), oscillator strengths (f), dipole moments (μ), resonance energies (RN) and ionization potentials (ID) are estimated. Thermodynamic parameters were computed from the thermal decomposition data.

Monitoring of HPLC profiles of selected polyphenolic compounds in sea buckthorn (Hippophaë rhamnoides L.) plant parts during annual growth cycle and estimation of their antioxidant potential

Abstract Presented work summarizes the data about polyphenolic profiles in various plant parts (leaves, shoots, berries) of sea buckthorn (Hippophaë rhamnoides L.) during the annual growth cycle. A reversed-phase high performance liquid chromatography method (RP-HPLC) coupled with diode-array detection (DAD) was optimized for determination of catechin, epicatechin, gallic acid, p-coumaric acid, caffeic acid, ferulic acid, rutin (quercetin 3-rutinoside) and quercitrin (quercetin 3-rhamnoside). The content of these polyphenolic compounds was monitored in extracts of sea buckthorn plant samples from April to October. The total antioxidant activity was determined using scavenging of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonate) cation radical (ABTS·+) and 1,1-diphenyl-2-picrylhydrazyl radical (DPPH·). The total content of polyphenols was estimated by conventional spectrophotometric method using Folin-Ciocalteu reagent. The monitoring of temporal changes of selected polyphenolic compounds by RP-HPLC showed that catechin, epicatechin and gallic acid were the most abundant analytes in annual green shoots and leaves, and their content varied significantly during the studied period.

Characterization of exopolymeric substances (EPS) produced by Aeromonas hydrophila under reducing conditions

The aim of this work was to investigate the production of extracellular polymeric substances (EPS) by Aeromonas hydrophila grown under anaerobic conditions. EPS composition was studied for planktonic cells, cells attached to carbon fibre supports using a soluble ferric iron source and cells grown with a solid ferric iron mineral (gossan). Conventional spectrophotometric methods, Fourier transform infrared (FTIR) and confocal laser scanning microscopy (CLSM) were used to determine the main components in the biofilm extracted from the cultures. The key EPS components were proteins, indicating their importance for electron transfer reactions. Carbohydrates were observed mostly on the mineral and contained terminal mannosyl and/or terminal glucose, fucose and N-acetylgalactosamine residues.

Direct Electrochemical Tyrosinase Biosensor based on Mesoporous Carbon and Co3O4 Nanorods for the Rapid Detection of Phenolic Pollutants

AbstractA novel tyrosinase (Tyr) biosensor based on a graphitized ordered mesoporous carbon/cobaltosic oxide nanorod (GMC/Co3O4) nanocomposite is developed for the rapid detection of phenolic pollutants. By applying the GMC/Co3O4 nanocomposite as an enzyme immobilization matrix, rapid direct electron transfer between Tyr and the electrode is achieved. The biosensor exhibits a wide linear response for catechol, ranging from 5.0×10−8 to 1.3×10−5 M, with a limit of detection down to 25 nM and a response time of less than 2 s. The sensitivity of the biosensor based on the GMC/Co3O4 nanocomposite (6.4 A M−1 cm−2) is higher than that of the biosensor based on GMC (5.0 A M−1 cm−2) or Co3O4 (3.5 A M−1 cm−2), which can be attributed to the synergistic effect of the GMC/Co3O4 nanocomposite. The biosensor is further used to systematically detect mixed phenolic samples (phenol, catechol, m‐cresol, p‐cresol, and 4‐chlorophenol) and real water samples. The biosensor‐based detection results for river water and tap water samples show outstanding average recovery (92.2–103.3 %) and relative standard deviations (0.9–7.8 %). In comparison with the conventional spectrophotometric method, the biosensor method is more rapid, sensitive, accurate, and convenient. Furthermore, the detection limit of the biosensor method is about 20 times lower than that of the spectrophotometric method. This novel biosensor is proven to be a promising alternative tool for the rapid and on‐site monitoring of environmental phenolic pollutants.

Analytical studies on the charge transfer complexes of loperamide hydrochloride and trimebutine drugs. Spectroscopic and thermal characterization of CT complexes

Charge transfer complexes of loperamide hydrochloride (LOP.HCl) and trimebutine (TB) drugs as electron donor with 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ), tetracyanoethylene (TCNE) and 7,7,8,8-tetracyanoquinodimethane (TCNQ) as π-acceptors in acetonitrile were investigated spectrophotometrically to determine the cited drugs in pure and dosage forms. The reaction gives highly coloured complex species which are measured spectrophotometrically at 460, 415 and 842nm in case of LOP.HCl and at 455, 414 and 842nm in case of TB using DDQ, TCNE and TCNQ reagents, respectively. The optimum experimental conditions have been studied carefully and optimized. Beer’s law was obeyed over the concentration ranges of 47.70–381.6, 21.50–150.5 and 10.00–100.0μgmL−1 for LOP.HCl and 37.85–264.9, 38.75–310.0 and 7.75–155.0μgmL−1 for TB using DDQ, TCNE and TCNQ reagents, respectively. Sandell sensitivity, standard deviation, relative standard deviation, limit of detection and quantification were calculated. The obtained data refer to high accuracy and precision of the proposed method. These results are also confirmed by inter and intra-day precision with percent recovery of 99.18–101.1% and 99.32–101.4% in case of LOP.HCl and 98.00–102.0% and 97.50–101.4% in case of TB using DDQ, TCNE and TCNQ reagents for intra- and inter-day, respectively. These data were compared with those obtained using official methods for the determination of the cited drugs. The stability constants of the CT complexes were determined. The final products of the reaction were isolated and characterized using FT-IR, 1H NMR, elemental analysis and thermogravimetric analysis (TG). The stoichiometry and apparent formation constant of the complexes formed were determined by applying the conventional spectrophotometric molar ratio method.

Contactless conductivity detection for screening myrosinase substrates by capillary electrophoresis

Myrosinase is a unique enzyme that catalyzes the hydrolysis of glucosinolates (GLS) to isothiocyanate (ITC), glucose and sulfate. Isothiocyanates display a diversified very interesting biological activity. In this study, capillary electrophoresis (CE) was used for the first time for evaluating myrosinase kinetics (maximum velocity Vmax and Michaelis–Menten constant Km) and to assess the affinity of a variety of substrates toward this enzyme.The pre-capillary approach was chosen since it is very simple to conduct. For this, the enzymatic reaction was performed in a micro-vial. The reaction mixture volume was of only 100μL and the incubation lasted only 5min at 37±1°C. Short-end injection of few tens of nanoliters (∼25nL) of the reaction mixture was performed which decreased analysis time without using any electroosmotic modifier. The sulfate produced was detected and quantified with a contactless capacitively coupled conductivity detector (C4D) allowing the evaluation of myrosinase kinetics.This study shows, that capillary electrophoresis with contactless conductivity detection can be very useful for monitoring myrosinase activity. Comparing to the conventional spectrophotometric method (1982), the CE method developed here is simple, automated, economic, rapid (incubation for few minutes) and robust. Results compared very well with those reported in literature using the conventional method. Moreover, the affinity of a variety of natural and synthetic glucosinolates toward this enzyme has been assessed for the first time.

Accuracy and Usefulness of the AVOXimeter 4000 as Routine Analysis of Carboxyhemoglobin

The measurement of blood carboxyhemoglobin (CO-Hb) is important to determine the cause of death. The AVOXimeter 4000 (AVOX), a portable CO-oximeter, has the advantages of a low purchase price and operating cost, ease of operation, and rapid results. Little information is available on the usefulness of AVOX in the forensic sample, and the previous study investigated only six samples. Therefore, in this study, we confirmed the usefulness of the AVOX through a comparison of its results with data previously obtained using the double wavelength spectrophotometric method in autopsies. Regression analysis was performed between CO-Hb levels measured by the AVOX and those measured by the conventional double wavelength spectrophotometric method in postmortem blood samples: a significant correlation was observed. This study suggests the usefulness of the AVOX to analyze postmortem blood, and the AVOX is suitable for routine forensic analysis and can be applied at the crime scene.

Quick determination of ammonia ions in water environment based on thymol color creating reaction

In this article, the thymol, one of phenolic derivative as color creative reagent and nitropruside as reaction catalyst were investigated for photometric and visual quick-test determination of ammonia in fresh and drinking water based on Berthelot (phenate) method. For this purpose, one test set should have maximum 2 to 3 mixed components solution. So the different solution preparation modes and reagents combination were studied. The investigation results showed that the 2 mixed solution mode was impossibly prepared because nitropruside was decomposed in strong oxidative solution such as hypochlorite and increased color intensity when mixed with thymol in strong alkaline solution by ageing. The 3 mixed solution mode was proposed and optimal components mixing was fount i. e. solution 1 is nitropruside in ammonia free water; solution 2 is hypochlorite in carbonate buffer at pH 10 and solution 3 is thymol and EDTA in 2M of sodium hydroxide. This 3 solution test set was used for ammonia quick determination. Following proposed test procedure, the full color creation takes less than 5 minutes and total time for one test is approximately 7 – 8 minutes. The ammonia concentration in different samples determined by photometric and visual method using 3 solution test set were compared with conventional spectrophotometric method. The measured values showed that analytical results were agreed each with other and the proposed test set could be used as test-kit for field measurement or common monitoring ammonia in fresh and drinking water. The maximum relative error is less than 25%.

A novel fluorescence method for determination of pFe3+

The fluorescence intensity of probe CP645 in the presence of iron and a competing ligand is associated with pFe(3+) of the competing ligand. A linear correlation was found to exist between the fluorescence and pFe(3+) values. The pFe(3+) values of well studied ligands calculated from the standard curve are consistent with reported values. This novel fluorescence method can be used to determine the pFe(3+) values of ligands which are difficult to measure using conventional spectrophotometric or potentiometric methods.

Direct quantification of creatinine in human urine by using isotope dilution extractive electrospray ionization tandem mass spectrometry

Urinary creatinine (CRE) is an important biomarker of renal function. Fast and accurate quantification of CRE in human urine is required by clinical research. By using isotope dilution extractive electrospray ionization tandem mass spectrometry (EESI–MS/MS) a high throughput method for direct and accurate quantification of urinary CRE was developed in this study. Under optimized conditions, the method detection limit was lower than 50μgL−1. Over the concentration range investigated (0.05–10mgL−1), the calibration curve was obtained with satisfactory linearity (R2=0.9861), and the relative standard deviation (RSD) values for CRE and isotope-labeled CRE (CRE-d3) were 7.1–11.8% (n=6) and 4.1–11.3% (n=6), respectively. The isotope dilution EESI–MS/MS method was validated by analyzing six human urine samples, and the results were comparable with the conventional spectrophotometric method (based on the Jaffe reaction). Recoveries for individual urine samples were 85–111% and less than 0.3min was taken for each measurement, indicating that the present isotope dilution EESI–MS/MS method is a promising strategy for the fast and accurate quantification of urinary CRE in clinical laboratories.