A highly sensitive micelle-enhanced synchronous spectrofluorimetric determination of the recently approved co-formulated drugs,bilastine and montelukast in pharmaceuticals and human plasma at nanogram levels

In this study, the simultaneous determination of bilastine and montelukast, two recently approved co-formulated antihistaminic medications, was accomplished using a quick, sensitive, environmentally friendly, and reasonably priced synchronous fluorescence spectroscopic approach for the first time. Enhancement of the method's sensitivity down to nanogram levels was achieved by the addition of sodium dodecyl sulfate (1.0% w/v) as a micellar system. According to the results, bilastine and montelukast's fluorescence was measured at 255.3 and 355.3 nm, respectively, using Δλ of 40.0 nm and distilled water as a green diluting solvent. With respect to the concentration ranges of bilastine (5.0–300.0 ng/ml) and montelukast (50.0–1000.0 ng/ml), the method showed excellent linearity (r ≥ 0.9998). The results showed that the suggested method is highly sensitive, with detection limits of 1.42 and 13.74 ng/ml for bilastine and montelukast, respectively. Within-run precisions (intra- and interday) per cent relative standard deviations (RSD) for both analytes were <0.59%. With high percentage recoveries and low percentage RSD values, the designed approach was successfully applied for the simultaneous estimation of the cited medications in their dosage form and human plasma samples. To evaluate the green profile of the suggested method, an analytical GREENNESS metric approach (AGREE) and green analytical procedure index (GAPI) metric tools were used. These two methods for evaluating greenness confirmed that the developed method met the highest number of green requirements, recommending its use as a green substitute for the routine analysis of the studied drugs. The proposed approach was validated according to ICHQ2 (R1) guidelines.     

Sensitive spectrofluorimetric determination of the prokinetic drug prucalopride succinate based on supramolecular aggregation approach with evaluation of method greenness: application to content uniformity test

The prokinetic drug, prucalopride (PCP) succinate, was determined using a new spectrofluorimetric approach with a highly sensitive, rapid, and simple procedure. The method exploited the enhancement of the inherent native fluorescence of PCP by micellar aggregation with sodium lauryl sulfate (SLS) as an anionic surfactant. Different factors that could affect the fluorescence intensity were carefully studied in order to achieve the maximal fluorescence signal. Measurement of the enhanced fluorescence was done at 354 nm after the excitation at 276 nm. The fluorescence intensity–concentration plot was rectilinear in the concentration range of 50–600 ng/ml with detection and quantitation limits of 13.9 and 42.1 ng/ml, respectively. The method underwent validation according to the International Council for Harmonisation criteria in order to assess its analytical performance, and promising results were achieved that proved the validity and reliability of the method. Furthermore, the method was employed effectively for the analysis of the cited drug in commercial pharmaceutical tablets.     

Spider diagram with greenness evaluation metrics for assessing the new synchronous spectrofluorimetric determination of bicalutamide and resveratrol in human plasma

A simple, selective, and eco-friendly synchronous fluorescence approach was introduced for the first time for the concurrent estimation of the anticancer combination therapy of bicalutamide and resveratrol. The method relies on measuring the synchronous fluorescence spectra of bicalutamide and resveratrol at 269 and 320 nm, respectively, using Δλ of 60 nm with ethanol as a green diluting solvent. The procedure was optimized, and the method was then fully validated. Excellent linearity (R² > 0.999) with very low detection limits (0.044 and 2.001 ng/ml) were obtained for both drugs, allowing for their analysis in human plasma. The green profile of the suggested approach was evaluated using the green solvents selecting tool (GSST), spider diagram for greenness index assessment, green analytical process index (GAPI), and Analytical GREEnness (AGREE) metric tools. These assessment metrics confirmed that the developed approach met the maximum number of green requirements, recommending its application as a green substitute for the regular analysis of the concerned drugs in human plasma. The simplicity of sample measurement enables and substantially accelerates the analysis, resulting in lower costs, enhanced procedure accuracy, and lower environmental effect.     

A novel sustainable second-derivative synchronous spectrofluorimetric method for the simultaneous determination of olmesartan medoxomil and rosuvastatin calcium in bulk and formulations: Integrated greenness and whiteness evaluation

The present research has established a quick and highly sensitive second-derivative synchronous fluorometric technique for the simultaneous quantification of a binary mixture of olmesartan medoxomil and rosuvastatin calcium. Simultaneously, the suggested approach was used to detect the synchronous fluorescence intensity of the cited drugs at Δ λ = 80 nm in ethanol to determine the concentrations of olmesartan medoxomil and rosuvastatin calcium at 265 and 240 nm, respectively. Various experimental conditions were tested, and each variable was analyzed and optimized. The calibration graphs were shown to be linear within ranges of 0.1–2.0 and 0.5–6.0 μg ml⁻¹ for each drug concentration, respectively. The newly developed Green Solvents Selecting Tool (GSST) was utilized to assess the solvent's sustainability. Furthermore, the proposed method was found to be environmentally friendly after being evaluated with three different tools [the Green Analytical Procedure Index (GAPI), the Analytical Greenness Metric (AGREE), and the Analytical Eco-Scale with Eco-score equal to 95]. The whiteness qualities were also studied using the Red–Green–Blue (RGB12) model, which was recently designed and showed a high score equal to 92.9. The proposed method’s good findings, as well as its ongoing sustainability, simplicity, and economy, stimulate its application in QC laboratories.     

Simple and sensitive spectrofluorimetric method for the determination of pregabalin in capsules through derivatization with fluorescamine

A new, simple and sensitive spectrofluorimetric method has been developed for the determination of pregabalin (PG) in capsules. The method is based on the reaction between pregabalin and fluorescamine in borate buffer solution of pH 10 to give a highly fluorescent derivative that is measured at 487 nm after excitation at 390 nm. The different experimental parameters affecting the development and stability of the reaction product were carefully studied and optimized. The fluorescence intensity concentration plot was rectilinear over the range of 0.01–0.3 µg mL^?1 with a lower detection limit of 0.0017 µg mL^?1 and limit of quantitation of 0.005 µg mL^?1. The developed method was successfully applied to the analysis of the drug in its commercial capsules. The mean percentage recovery of PG in its capsule was 99.93±1.24 (n = 3). Statistical comparison of the results with those of the comparison method revealed good agreement and proved that there was no significant difference in the accuracy and precision of the two methods. A proposed reaction pathway was postulated. Copyright © 2010 John Wiley & Sons, Ltd.     

Safety of milk and milk derivatives in relation to BSE: the lactoferrin example

Bovine Spongiform Encephalopathy (BSE) belongs to Transmissible Spongiform Encephalopathies (TSEs) or Prion diseases. BSE is a feed borne infection of cattle. Epidemiological and laboratory data suggest that the BSE infectious agent is responsible for the variant form of Creutzfeldt-Jakob Disease (vCJD) and that the oral route is the most plausible way of infection. Therefore there is concern that the BSE agent can be transmitted to humans by biological materials (i.e. meat products, blood, milk) from susceptible BSE animal species (mostly cows but possibly, sheep and goats). Lactoferrin (LF) can be produced by purification from large volumes of cow's milk or whey. Therefore, a potential BSE risk for milk and milk products needs to be evaluated by risk assessment. The Committee for proprietary Medicinal Products--CPMP of the European Commission and the WHO have categorized risk tissues from TSE susceptible ruminant species in different classes in relation to the BSE risk for medicinal products. Milk, colostrum, and tissues of the mammary gland have been classified in the category of no detectable infectivity. A secondary contamination of milk can be virtually excluded (i.e. milk is taken from living animals). In the light of current scientific knowledge and irrespective of the geographical origin, milk and milk derivatives (e.g. lactoferrin, lactose) are unlikely to present any risk of TSE contamination provided that milk is sourced from healthy animals in the same conditions as milk collected from human consumption. So the risk of milk and milk derivatives in relation to BSE is negligible.     

A novel spectrofluorimetric method for the determination of calcitonin in ampules through derivatization with fluorescamine

In this study, a simple, sensitive and selective spectroflourimetric method has been developed for the determination of salmon calcitonin (sCT) in ampules. The method is based on the reaction between sCT and fluorescamine at pH 8.5 in borate buffer, resulting in a highly fluorescent derivative. Fluorescence of derivatized sCT solutions was measured by setting the excitation and emission monochromators and slit widths to 390, 484 and 10 nm, respectively. Sevaral derivatization parameters were optimized. A calibration graph was constructed using standard solutions of the derivatized calcitonin in the range 0.5–6.0 µg/mL. Limit of detection and limit of quantification values were determined to be 0.124 and 0.372 µg/mL, respectively. The proposed method was successfully applied for the determination of sCT in commercially available ampules. High recovery values (101.0–102.0 %), and a low relative standard deviation (RSD %) value (5.3–5.4) proved the accuracy and precision of the proposed method. An isocratic reversed‐phase high‐performance liquid chromatographic (HPLC) method, as a reference, was also developed for the determination of sCT. A reversed‐phase Nucleosil® C₁₈ column (250 mm × 4.6 mm i.d., 10 µm particle size, 120 Å pore size) was used and the detector was set at 210 nm. Statistical comparison of the results of the two methods showed clearly that there was no significant difference between them. Copyright © 2012 John Wiley & Sons, Ltd.     

Characterisation and quantification of protein oxidative modifications and amino acid racemisation in powdered infant milk formula

Modification of proteins in infant milk formula (IF) is of major concern to the dairy industry and consumers. Thermal treatment is required for microbiological safety, but heat, light, metal-ions and other factors may induce oxidative damage, and be a health risk. In this study protein modifications in IFs were quantified. IFs contained both reducible (disulphide) and non-reducible (di-tyrosine, lanthionine, lysinoalanine) protein cross-links. Dehydroalanine and the cross-linked species lanthionine and lysinoalanine were detected. Protein carbonyls were detected predominantly on high molecular mass materials. Oxidation products of phenylalanine (m-tyrosine), tryptophan (N-formylkynurenine, kynurenine, 3-hydroxykynurenine), tyrosine (di-tyrosine) and methionine (methionine sulphoxide) were detected, consistent with amino acid modification. Higher levels of most of the markers of protein modification were present in the hydrolysed protein brand, when compared to the conventional IF samples, indicative of increased damage during additional processing. Significant levels of racemised (D-) amino acids were present. These data indicate that amino acids in proteins in IFs are modified to a significant extent during manufacture, with hydrolysed IF being particularly prone.     

Development of sensitive spectrofluorimetric and spectrophotometric methods for the determination of duloxetine in capsule and spiked human plasma

A new, sensitive and selective spectrofluorimetric method has been developed for the determination of duloxetine (DLX) in capsule and spiked human plasma. DLX, as a secondary amine compound, reacts with 7‐chloro‐4‐nitrobenzofurazon (NBD‐Cl), a highly sensitive fluorogenic and chromogenic reagent used in many investigations. The method is based on the reaction between the drug and NBD‐Cl in borate buffer at pH 8.5 to yield a highly fluorescent derivative that is measured at 523 nm after excitation at 478 nm. The fluorescence intensity was directly proportional to the concentration over the range 50–250 ng/mL. The reaction product was also measured spectrophotometrically. The relation between the absorbance at 478 nm and the concentration is rectilinear over the range 1.0–12.0 µg/mL. The methods were successfully applied for the determination of this drug in pharmaceutical dosage form. The spectrofluorimetric method was also successfully applied to the determination of duloxetine in spiked human plasma. The suggested procedures could be used for the determination of DLX in pure form, capsules and human plasma being sensitive, simple and selective. Copyright © 2014 John Wiley & Sons, Ltd.     

Development and validation of a sensitive spectrofluorimetric method for the determination of amoxapine in human plasma and urine

A selective and sensitive spectrofluorimetric method was developed and validated for the determination of amoxapine in human plasma and urine. The developed method is based on labeling with 5‐dimethylaminonaphthalene‐1‐sulfonyl chloride (dansyl chloride) and monitoring at 397 nm (excitation)/514 nm (emission). The method was validated for linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, recovery and robustness. The calibration curves were linear over a concentration range of 250–2500 and 50–1250 ng/mL for plasma and urine, respectively. The LOD values were calculated to be 13.31 and 13.17 ng/mL for plasma and urine, respectively. The proposed method was applied to study of amoxapine in human plasma and urine. Copyright © 2013 John Wiley & Sons, Ltd.     

In vivo digestomics of milk proteins in human milk and infant formula using a suckling rat pup model

Human milk is the optimal mode of infant feeding for the first several months of life, and infant formulas serve as an alternative when breast-feeding is not possible. Milk proteins have a balanced amino acid composition and some of them provide beneficial bioactivities in their intact forms. They also encrypt a variety of bioactive peptides, possibly contributing to infant health and growth. However, there is limited knowledge of how milk proteins are digested in the gastrointestinal tract and bioactive peptides are released in infants. A peptidomic analysis was conducted to identify peptides released from milk proteins in human milk and infant formula, using a suckling rat pup model. Among the major milk proteins targeted, α-lactalbumin and β-casein in human milk, and β-lactoglobulin and β-casein in infant formula were the main sources of peptides, and these peptides covered large parts of the parental proteins’ sequences. Release of peptides was concentrated to specific regions, such as residues 70–92 of β-casein in human milk, residues 39–55 of β-lactoglobulin in infant formula, and residues 57–96 and 145–161 of β-CN in infant formula, where resistance to gastrointestinal digestion was suggested. In the context of bioactive peptides, release of fragments containing known bioactive peptides was confirmed, such as β-CN-derived opioid and antihypertensive peptides. It is therefore likely that these fragments are of biological significance in neonatal health and development.     

Highly sensitive spectrofluorimetric method for rapid determination of orciprenaline in biological fluids and pharmaceuticals

Orciprenaline sulphate (ORP) is a direct‐acting sympathomimetic with mainly beta‐adrenoceptor stimulant activity. It is used as a bronchodilator in the management of reversible airway obstruction. For the first time, a rapid highly sensitive spectrofluorimetric method is described that is relied on measuring the fluorescence spectra of ORP at acidic pH and without addition of any chemical reagents. The relative fluorescence intensity was measured at 310 nm and after excitation at 224 nm. ORP native fluorescence was calibrated in both water and acetonitrile as diluting solvents. The method was designed to estimate the drug in miscellaneous matrices with high accuracy and precision. Linear ranges of calibration curves were 30.0–400.0 ng/ml and 10.0–240.0 ng/ml in water and acetonitrile, respectively. The detection limits were calculated and reached as low as 3.3 and 3.1 ng/ml, respectively, representing the ultra‐sensitivity of the proposed method. This result permitted application of this method for spiked human plasma and urine and was used as a preliminary investigation with good percentage recovery (89.4–106.8%). The application was further extended to analyse ORP in its pharmaceutical formulations. The method was validated in compliance with International Council of Harmonization (ICH) Guidelines.     

A new spectrofluorimetric method for determination of trace amounts histamine in human urine and serum

A new spectrofluorimetric method was developed for the determination of trace amounts of histamine in human urine and serum samples. In NaAc–HAc buffer solution of pH 4.0, histamine can react with the acetylacetone–formaldehyde system to produce a fluorescent derivative which emits yellow‐green fluorescence at 476 nm, according to the Hantzsch reaction, and the enhanced fluorescence intensity is in proportion to the concentration of histamine. Optimum conditions for the determination of histamine were also investigated. The dynamic range and detection limit for the determination of histamine is 5.96 × 10^–8–1.50 × 10^–5 mol/L and 4.35 × 10^–8mol/L, respectively. This method is practical and can be successfully applied to determination of histamine in human urine and serum samples. A proposal of the reaction pathway is suggested. Copyright © 2009 John Wiley & Sons, Ltd.     

Effect of environmental stresses on the sensitivity of Enterobacter sakazakii in powdered infant milk formula to gamma radiation

Aim:  To evaluate the effect of starvation, heat, cold, acid, alkaline, chlorine and ethanol stresses on the resistance of Enterobacter sakazakii in powdered infant milk formula (PIMF) towards gamma radiation.
Methods and Results:  Stressed cells of E. sakazakii ATCC 51329 and four other food isolate strains were mixed individually with PIMF, kept overnight at room temperature, and then exposed to gamma radiation up to 7·5 kGy. The D ₁₀-values were determined using linear regression and for the stressed E. sakazakii strains these values ranged from 0·82 to 1·95 kGy.
Conclusions:  Environmental stresses did not significantly change the sensitivity of most E. sakazakii strains to ionizing radiation.
Significance and Impact of the Study:  Data obtained established that most forms of environmental stress are unlikely to significantly enhance the resistance of E. sakazakii strains to lethal, low dose irradiation treatment.     

Green sensitive spectrofluorimetric determination of pemetrexed as antineoplastic drug: Application in pharmaceutical dosage forms and spiked human plasma

A recent antineoplastic medication is pemetrexed, this medicine is now being developed and produced on a large scale, thus approaches for quality control are urgently needed. Spectrofluorimetric guidelines for the simple estimation of pemetrexed were validated. Pemetrexed's assay depends on observations of its native fluorescence at wavelengths 275/450 nm and pH 4. The proposed approach was also used to identify the examined drug in both its formulation and in human plasma that had been spiked.     

Two green and sensitive spectrofluorimetric approaches for determination of Ambroxol and guaifenesin in their single and combined pharmaceutical formulations

Ambroxol hydrochloride (AMX) and guaifenesin (GFN) are approved drugs utilized to treat coughs through their potent mucolytic and expectorant properties. Due to their massive, combined administration in many illnesses, there is a persistent need for their concurrent estimation in different pharmaceutical formulations. Two sensitive, environmentally friendly spectrofluorimetric methods were developed. AMX was determined using the first method (I) without interference from GFN. This method depends on the quenching of Erythrosine B (EB) native fluorescence at 552 nm after excitation at 527 nm due to the formation of a non-fluorescent AMX-EB ion-pair complex in Britton–Robinson buffer (BRB) solution pH (3.5). The concentration plot is linear over the 0.25–5.0 μg/mL range, with a mean percent found value of 99.74%. Method (II) depends on measuring the native fluorescence of aqueous GFN solution at two analytical wavelengths, either 300 or 600 nm, after excitation at 274 nm. Relative fluorescence intensity (RFI)–concentration plots are linear over the ranges of 0.02–0.5 and 0.1–2.0 μg/ml, with mean percent found at 99.96% and 99.91% at dual wavelengths, respectively. The proposed methods were successfully applied to assay both drugs in raw materials and different single and combined pharmaceutical formulations. These methods have been thoroughly validated following International Committee on Harmonisation (ICH) guidelines. National Environmental Methods Index, Analytical Eco-Scale, and Green Analytical Procedure Index were used to prove greenness, thereby enhancing their applicability. The proposed techniques provide straightforward, precise, and cost-effective solutions for routine formulation analysis in quality control laboratories.     

Microenvironment improvement protocol for the sensitive spectrofluorimetric determination of an hepatitis C virus antiviral (Simeprevir): application to human plasma

Simeprevir (SPV) is a powerful antihepatitis C virus agent that was newly introduced into the pharmaceutical market. We here established and validated an easy, simple, and sensitive spectrofluorimetric method for its estimation at λ_em 427 nm (λ_ex 337 nm). The suggested procedure was based on two times enhancement in the original emission of SPV through modifying its microenvironment in buffered aqueous solution by adding Triton X‐100. The relationship between the concentration of SPV and the observed fluorescence intensity was linear in the range 0.06–1.0 μg ml^?1 with a correlation coefficient of 0.9997. The limits of detection and quantitation were 21 and 64 ng ml^?1, respectively. The present method was effectively applied to quantify SPV content in pharmaceutical tablets and human plasma spiked with the drug with no interference from tablet excipients or plasma components.     

Highly sensitive spectrofluorimetric method for the determination of the genotoxic methylglyoxal in glycerol-containing pharmaceuticals and dietary supplements

Methylglyoxal (MGO) is a genotoxic α-dicarbonyl compound. Recently, it was found to be formed in glycerol preparations during storage through auto-oxidation. A simple fluorimetric determination of the carcinogenic degradation product of glycerol, MGO, was developed and validated. The proposed method is based on the derivatization of MGO with 4-carbomethoxybenzaldehyde (CMBA) and ammonium acetate to yield a fluorescent imidazole derivative that can be measured at 415 nm after excitation at 322 nm. The optimized conditions were determined to be 0.2 M CMBA, 1.0 M ammonium acetate and a reaction time of 40 min at 90°C using ethanol as diluting solvent. The linear range was 10.0–200.0 ng/ml. Detection and quantification limits were 2.22 and 6.72 ng/ml, respectively. The proposed method was validated according to International Council for Harmonisation (ICH) guidelines and compared with the reported method and no significant difference was found. It was successfully applied for the determination of MGO in six different glycerol-containing pharmaceutical preparations and dietary supplements.     

A new spectrofluorimetric method for determination of losartan potassium in rabbit plasma and its application to pharmacokinetic study

A new spectrofluorimetric method to determine losartan potassium (LP) in rabbit plasma is described. The method was based on measuring the native fluorescence of LP in acidic medium. Optimum excitation and emission wavelengths were found to be 248 nm and 410 nm, respectively, in methanol that was diluted with a sulfurous acid solution LP was extracted from rabbit plasma by methyl‐tertiary‐butyl‐ether in acidic media and then back extracted with NaOH. The calibration curves were linear between 0.025 and 0.5 µg/mL with a lower limit of detection 0.004 µg/mL. Precision and accuracy values of the method were calculated as lower than 4.97% and ± 5.68, respectively and the recovery of LP from rabbit plasma was higher than 91.1%. In addition, stability studies of LP in rabbit plasma were carried out and demonstrated its good stability at − 20 °C and at room temperature. The developed and validated method was successfully applied for estimating the pharmacokinetic parameters of LP following oral administrations of a single 10 mg LP/kg to rabbits and it could be concluded that the method can be applied to clinical trials. Copyright © 2014 John Wiley & Sons, Ltd.