NIR-Fluorescent Multidye Silica Nanoparticles with Large Stokes Shifts for Versatile Biosensing Applications.

We have synthesized and characterized of a series of single and multidye copolymerized nanoparticles with large to very large Stokes shifts (100 to 255 nm) for versatile applications as standalone or multiplexed probes in biological matrices. Nanoparticles were prepared via the Stöber method and covalently copolymerized with various combinations of three dyes, including one novel aminocyanine dye. Covalently encapsulated dyes exhibited no significant leakage from the nanoparticle matrix after more than 200 days of storage in ethanol. Across multiple batches of nanoparticles with varying dye content, the average yields and average radii were found to be highly reproducible. Furthermore, the batch to batch variability in the relative amounts of dye incorporated was small (relative standard deviations <2.3%). Quantum yields of dye copolymerized nanoparticles were increased 50% to 1000% relative to those of their respective dye-silane conjugates, and fluorescence intensities were enhanced by approximately three orders of magnitude. Prepared nanoparticles were surface modified with polyethylene glycol and biotin and bound to streptavidin microspheres as a proof of concept. Under single wavelength excitation, microsphere-bound nanoparticles displayed readily distinguishable fluorescence signals at three different emission wavelengths, indicating their potential applications to multicolor sensing. Furthermore, nanoparticles modified with polyethylene glycol and biotin demonstrated hematoprotective qualities and reduced nonspecific binding of serum proteins, indicating their potential suitability to in vivo imaging applications.

A Comparative Study of Fluorescein Isothiocyanate-Encapsulated Silica Nanoparticles Prepared in Seven Different Routes for Developing Fingerprints on Non-Porous Surfaces.

Preparation of fluorescein isothiocyanate (FITC)-encapsulated silica nanoparticles (F-SiNPs) via seven different approaches to be used as developing agents for fingerprints detection is presented in this report. In this study, the suitability of each synthesis route toward incorporation of the selected fluorophore into silica matrix and its efficiency in fingerprints detection were systematically studied. The composition of the particles was designed to examine the hydrophobic and dipole-dipole interactions between the silicate backbone and both of the fluorescent reporter molecules and the fingerprint residues. F-SiNPs were prepared with two conventional sol-gel approaches; the Stöber method and the water in oil reverse microemulsion (WORM) method. The alkoxysilane precursor, tetraethoxyorthosilicate (TEOS) and its binary mixtures with phenyltriethoxysilane (PTEOS) or 3-aminopropyl triethoxysilane (APTES) have been used in preparing the F-SiNPs to study the effect of nanoparticles composition on fingerprints development. In addition, FITC was conjugated with APTES so it can be covalently bonded to the silica matrix and to be compared with non-covalently FITC-doped SiNPs. Moreover, the enhancement effect of introducing polyvinylpyrrolidone (PVP) onto the surface of the less hydrophobic F-SiNP on fingerprints detection on different non-porous surfaces was also investigated. The mean diameters of the F-SiNPs were between 4.1 ± 0.6 and 110.4 ± 31.1 nm as obtained from the TEM size measurement for the nanoparticles prepared by the WORM and Stöber methods, respectively. The obtained results clearly highlight the advantages of using a mixture of TEOS and PTEOS alkoxysilane precursors in preparing F-SiNPs with remarkable encapsulation efficiency and clear detection of fingerprints due to efficient embedding of the fluorophore inside the silica network even without conjugation. It was also observed that both the Stöber and WORM methods can be used in preparing the F-SiNPs developing agents and that PVP coated particles did not show any significant enhancement in fingerprints visualization.

Preparation of fluorescently labeled silica nanoparticles using an amino acid-catalyzed seeds regrowth technique: Application to latent fingerprints detection and hemocompatibility studies.

The efficiency of an amino acid catalyzed seed regrowth technique (ACSRT) in synthesizing twelve fluorescently labeled core-shell silica nanoparticles (FLSNPs) with tunable sizes, tailored hydrophobicity, low polydispersity as well as high labeling efficiency and minimized dye leakage using different combinations of organosilicate monomers and fluorophores have been systematically investigated in this report. The utilization of some of these FLSNPs in some applications that are facilitated by hydrophobicity such as developing and visualizing latent fingerprints (LFPs) on different surfaces was also investigated. The non-specific binding affinity of the developed nanoparticles to human serum albumin (HSA) and immunoglobulin G (IgG) has also been studied. Fluorescein, fluorescein isothiocyanate and its more hydrophilic butenamine derivative (WA6) have been used in this study. Also, the alkoxysilane precursor, tetraethoxyorthosilicate (TEOS) and its binary mixture with phenyltriethoxysilane (PTEOS) or 3-aminopropyl triethoxysilane (APTES) have been used in preparing the FLSNPs with tailored compositions for the core and shell of the nanoparticles. The mean diameters of the PTEOS-coated FLSNPs were between 33.4±5.9 and 42.2±10.8 nm as shown by the SEM measurements. The obtained results highlight the advantages of having a hydrophobic surface along with proper selection of the monomers forming the core to match the properties of the fluorescent reporters for clear detection of LFPs even using dyes of low hydrophobicity such as fluorescein and WA6. Furthermore, some of the developed FLSNPs were compared with bare silica nanoparticles in terms of nonspecific protein adsorption and hemolysis. The obtained results proved that the selected FLSNPs had a superior hemocompatibility in comparison with bare silica nanoparticles. These FLSNPs could also be used in some bio-related and diagnostic applications such as immunoassays and cell imaging purposes.

Simultaneous Determination of Aliskiren Hemifumarate, Amlodipine Besylate and Hydrochlorothiazide in Spiked Human Plasma Using UPLC-MS/MS.

A sensitive UPLC-MS/MS method was developed and validated for simultaneous estimation of aliskiren hemifumarate (ALS), amlodipine besylate (AML) and hydrochlorothiazide (HCZ) in spiked human plasma using valsartan as an internal standard (IS). Liquid-liquid extraction was used for purification and pre-concentration of analytes. The mobile phase consisted of 0.1% formic acid in ammonium acetate buffer (0.02 M, pH 3.5) and methanol (25:75, v/v), flowing through XBridge BEH (50 × 2.1 mm ID, 5 µm) C18 column, at a flow rate of 0.6 mL min(-1). Multiple reaction monitoring (MRM) transitions were measured using an electrospray source in the positive ion mode for ALS and AML, whereas HCZ and IS were measured in negative ion mode. Validation of the method was performed as per US-FDA guidelines with linearity in the range of 2.0-400.0, 0.3-25.0 and 5.0-400.0 ng mL(-1) for ALS, AML and HCZ, respectively. In human plasma, ALS, AML and HCZ were stable for at least 1 month at -70 ± 5°C and for at least 6 h at ambient temperature. After extraction from plasma, the reconstituted samples of ALS, AML and HCZ were stable in the autosampler at ambient temperature for 6 h. The LC-MS/MS method is suitable for bioequivalence and pharmacokinetic studies of this combination.

Stability-indicating RP-LC method for determination of azilsartan medoxomil and chlorthalidone in pharmaceutical dosage forms: application to degradation kinetics.

A RP-LC method was developed and validated for simultaneous determination of the active components, azilsartan medoxomil (AZL) and chlorthalidone (CLT), in their novel antihypertensive combined recipe. The chromatographic separation was achieved on an Eclipse XDB-C18 (4.6 × 150 mm, 5 µm) column using a mobile phase consisting of methanol/potassium hydrogen phosphate buffer (pH 8, 0.05 M) (40:60, v/v) in isocratic mode. The flow rate was maintained at 0.8 mL min(-1) at ambient temperature. Detection was carried out at 210 nm. The method was validated according to the ICH guidelines. Linearity, accuracy, and precision were satisfactory over the concentration range of 5.0-50.0 and 2.5-25.0 µg mL(-1) for AZL and CLT, respectively (r (2) = 0.9999). LODs for AZL and CLT were 0.90 and 0.32 µg mL(-1), whereas LOQs were 2.72 and 0.98 µg mL(-1), respectively. Both drugs were subjected to forced degradation studies under hydrolysis (neutral, acidic, and alkaline), oxidative, and photolytic extensive stress conditions. The proposed method is stability indicating by the resolution of the investigated drugs from their degradation products. Moreover, the kinetics of the acidic degradation of AZL as well as the kinetics of the alkaline degradation of CLT were investigated. Arrhenius plots were constructed and the apparent first-order rate constants, half-life times, shelf-life times, and the activation energies of the degradation processes were calculated. The method was successfully applied for the determination of the studied drugs simultaneously in their coformulated tablet. The developed method is specific and stability indicating for the quality control and routine analysis of the cited medications in their pharmaceutical preparations.

Spectrophotometric and spectrofluorimetric studies on azilsartan medoxomil and chlorthalidone to be utilized in their determination in pharmaceuticals.

The recently approved angiotensin II receptor blocker, azilsartan medoxomil (AZL), was determined spectrophotometrically and spectrofluorimetrically in its combination with chlorthalidone (CLT) in their combined dosage form. The UV-spectrophotometric technique depends on simultaneous measurement of the first derivative spectra for AZL and CLT at 286 and 257 nm, respectively, in methanol. The spectrofluorimetric technique depends on measurement of the fourth derivative of the synchronous spectra intensities of AZL in presence of CLT at 298 nm in methanol. The effects of different solvents on spectrophotometric and spectrofluorimetric responses were studied. For, the spectrofluorimetric study, the effect of pH and micelle-assisted fluorescence enhancement were also studied. Linearity, accuracy, and precision were found to be satisfactory over the concentration ranges of 8-50 µg mL(-1) and 2-20 µg mL(-1) for AZL and CLT, respectively, in the spectrophotometric method as well as 0.01-0.08 µg mL(-1) for AZL in the spectrofluorimetric method. The methods were successfully applied for the determination of the studied drugs in their co-formulated tablets. The developed methods are inexpensive and simple for the quality control and routine analysis of the cited drugs in bulk and in pharmaceuticals.

Steady-state and synchronous spectrofluorimetric methods for simultaneous determination of aliskiren hemifumarate and amlodipine besylate in dosage forms.

Aliskiren hemifumarate (ALS) and amlodipine besylate (AML) were simultaneously determined by two different spectrofluorimetric techniques. The first technique depends on direct measurement of the steady-state fluorescence intensities of ALS and AML at 313 nm and 452 nm upon excitation at 290 and 375 nm, respectively, in a solvent composed of methanol and water (10: 90, v/v). The second technique utilizes synchronous fluorimetric quantitative screening of the emission spectra of ALS and AML at 272 and 366 nm, respectively using Δλ of 97 nm. Effects of different solvents and surfactants on relative fluorescence intensity were studied. The method was validated according to ICH guidelines. Linearity, accuracy and precision were found to be satisfactory in both techniques over the concentration ranges of 1-15 and 0.4-4 µg/mL for ALS and AML, respectively. In the first technique, limit of detection and limit of quantification were estimated and found to be 0.256 and 0.776 µg/mL for ALS as well as 0.067 and 0.204 µg/mL for AML, respectively. Also, limit of detection and limit of quantification were calculated in the synchronous method and found to be 0.293 and 0.887 µg/mL for ALS as well as 0.034 and 0.103 µg/mL for AML, respectively. The methods were successfully applied for the determination of the two drugs in their co-formulated tablets. The results were compared statistically with reference methods and no significant difference was found. The developed methods are rapid, sensitive, inexpensive and accurate for the quality control and routine analysis of the cited drugs in bulk and in pharmaceutical preparations without pre-separation.

Simultaneous determination of aliskiren hemifumarate, amlodipine besylate, and hydrochlorothiazide in their triple mixture dosage form by capillary zone electrophoresis.

A novel, specific, reliable, and accurate capillary zone electrophoretic method was developed and validated for the simultaneous determination of aliskiren hemifumarate, amlodipine besylate, and hydrochlorothiazide in their triple mixture dosage form. Separation was carried out in a fused-silica capillary (57.0 cm total length and 50.0 cm effective length, 75.6 µm internal diameter) by applying a potential of 17 kV and a running buffer consisting of 40 mM phosphate buffer at pH 6.0 with UV detection at 245 nm. The method was suitably validated with respect to specificity, linearity, LOD, and LOQ, accuracy, precision, and robustness. The method showed good linearity in the ranges 1-10, 2.5-25, and 30-300 µg/mL with LODs of 0.11, 0.33, and 5.83 µg/mL for amlodipine besylate, hydrochlorothiazide, and aliskiren hemifumarate, respectively. The proposed method was successfully applied for the analysis of the studied drugs in their coformulated tablets. The results of the proposed method were statistically compared with those obtained by the RP-HPLC reference method revealing no significant differences in the performance of the methods regarding accuracy and precision.

Synchronized separation of seven medications representing most commonly prescribed antihypertensive classes by using reversed-phase liquid chromatography: Application for analysis in their combined formulations.

A reversed-phase high-performance liquid chromatography method was developed for the simultaneous determination of the diuretic, hydrochlorothiazide, along with six drugs representing the most commonly prescribed antihypertensive pharmacological classes such as atenolol, a selective ß1 blocker, amlodipine besylate, a calcium channel blocker, moexipril hydrochloride, an angiotensin-converting-enzyme inhibitor, valsartan and candesartan cilexetil, which are angiotensin II receptor blockers, and aliskiren hemifumarate, a renin inhibitor, using irbesartan as an internal standard. The chromatographic separation was achieved using acetonitrile/sodium phosphate dibasic buffer (0.02 M, pH 5.5) at a flow rate of 1 mL/min in gradient elution mode at ambient temperature on a stationary phase composed of an Eclipse XDB-C18 (4.6 × 150 mm, 5 µm) column. UV detection was carried out at 220 nm. The method was validated according to ICH guidelines. Linearity, accuracy, and precision were satisfactory over the concentration ranges of 2-40 µg/mL for hydrochlorothiazide and candesartan cilexetil, 20-120, 10-160, 5-40, 20-250, and 5-50 µg/mL for atenolol, valsartan, moexipril hydrochloride, aliskiren hemifumarate, and amlodipine besylate, respectively. The method was successfully applied for the determination of each of the studied drugs in their combined formulations with hydrochlorothiazide. The developed method is suitable for the quality control and routine analysis of the cited drugs in their pharmaceutical dosage forms.

Simultaneous determination of sitagliptin and metformin in pharmaceutical preparations by capillary zone electrophoresis and its application to human plasma analysis.

A novel, quick, reliable and simple capillary zone electrophoresis CZE method was developed and validated for the simultaneous determination of sitagliptin (SG) and metformin (MF) in pharmaceutical preparations. Separation was carried out in fused silica capillary (50.0 cm total length and 43.0 cm effective length, 49 µm i.d.) by applying a potential of 15 KV (positive polarity) and a running buffer containing 60 mM phosphate buffer at pH 4.0 with UV detection at 203 nm. The samples were injected hydrodynamically for 3 s at 0.5 psi and the temperature of the capillary cartridge was kept at 25 °C. Phenformin was used as internal standard (IS). The method was suitably validated with respect to specificity, linearity, limit of detection and quantitation, accuracy, precision, and robustness. The method showed good linearity in the ranges of 10-100 µg/mL and 50-500 µg/mL with limits of detection of 0.49, 2.11 µg/mL and limits of quantification of 1.48, 6.39 µg/mL for SG and MF, respectively. The proposed method was successfully applied for the analysis of the studied drugs in their synthetic mixtures and co-formulated tablets without interfering peaks due to the excipients present in the pharmaceutical tablets. The method was further extended to the in-vitro determination of the two drugs in spiked human plasma. The estimated amounts of SG/MF were almost identical with the certified values, and their percentage relative standard deviation values (% R.S.D.) were found to be ≤1.50% (n = 3). The results were compared to a reference method reported in the literature and no significant difference was found statistically.

The solvatochromic effects of side chain substitution on the binding interaction of novel tricarbocyanine dyes with human serum albumin.

The effects of solvatochromism on protein-ligand interactions have been studied by absorbance and near-infrared laser induced fluorescence (NIR-LIF) spectroscopy. The utility of three novel classes of cyanine dyes designed for this purpose illustrates that the affinity interactions of ligands at the hydrophobic binding pockets of Human Serum Albumin (HSA) are not only dependent on the overall hydrophobic characteristics of the molecules but are highly influenced by the size of the ligands as well. Whereas changes to the chromophore moiety exhibited slight to moderate changes to the hydrophobic nature of these molecules, substitution at the alkyl indolium side chain has enabled us to vary the binding affinity towards serum albumin. Substitution at the indolium side chain among an ethyl to butyl group results in improved binding characteristics and an almost three-fold increase in affinity constant. In addition, replacement of the ethyl side chain with a phenylpropyl group also yielded unique solvotachromic patterns such as increased hydrophobicity and subsequent biocompatibility with the HSA binding regions. Ligand interaction was however inhibited by steric hindrance associated with the bulky phenyl ring system thus affecting the increased binding that could be realized from the improved hydrophobic nature of the molecules. This characteristic change in binding affinity is of potential interest to developing a methodology which reveals information on the hydrophobic character and steric specificity of the binding cavities.

The effect of varying short-chain alkyl substitution on the molar absorptivity and quantum yield of cyanine dyes.

The effect of varying short-chain alkyl substitution of the indole nitrogens on the spectroscopic properties of cyanine dyes was examined. Molar absorptivities and fluorescence quantum yields were determined for a set of pentamethine dyes and a set of heptamethine dyes for which the substitution of the indole nitrogen was varied. For both sets of dyes, increasing alkyl chain length resulted in no significant change in quantum yield or molar absorptivity. These results may be useful in designing new cyanine dyes for analytical applications and predicting their spectroscopic properties.

A new strategy utilizing electrospray ionization-quadrupole ion trap mass spectrometry for the qualitative determination of GnRH peptides.

Numerous forms of the neurotransmitter GnRH have been discovered in vertebrates and invertebrates. Methods used for identification of these peptides are laborious and often require the application of multiple, confirmatory techniques. In this study, we investigate whether HPLC-MS/MS and de novo sequencing techniques applied to whole peptide analysis can provide a simpler approach to GnRH characterization. Experiments were performed with six GnRH forms (chicken I, chicken II, lamprey III, mammalian, salmon and seabream) to determine whether MS/MS spectra would be dominated by proline-directed fragmentation to the detriment of obtaining sufficient fragmentation for sequencing. While the expected b8 fragment was prominent, sufficient ion series were obtained for the six GnRH peptides to provide sequence identification. On the basis of the patterns observed for six model peptides, similar fragmentation patterns are expected for other GnRH forms. To confirm the applicability of the method, extracts from Sprague-Dawley rat brains were examined. These experiments confirm the presence of mammalian GnRH and a posttranslationally modified form of mammalian GnRH, hydroxyproline9 GnRH, in Sprague-Dawley rat brains and demonstrate that ESI-MS/MS techniques provide a valuable addition to existing qualitative methods.

Spectroscopic study of a novel bis(heptamethine cyanine) dye and its interaction with human serum albumin.

A newly synthesized near-infrared (NIR) bis(heptamethine cyanine) dye 7 was evaluated for its utility as a non-covalent label for proteins. This dye forms inter- and intramolecular H-aggregates in polar solvents, even at very low concentrations. The intramolecular dimeric form of the dye can be described as a clam-shell complex with two interacting hydrophobic carbocyanine moieties. In this intramolecular H-aggregate, the chromophore has a low extinction coefficient and low fluorescence quantum yield. In aqueous solution, in the absence of human serum albumin (HSA), dye 7 has characteristic absorption bands at 792 and 435 nm, and its fluorescent emission is significantly diminished in comparison to that in methanol or when compared to its monomeric equivalent 5. Dye 7 seems to be more advantageous than its monomeric counterpart 5 as a non-covalent label for biomolecules. Upon addition of HSA, the H and D bands are decreased and the monomeric band is increased, with concomitant increase in fluorescence intensity, suggesting that clam-shell H-aggregates open up in the complex with HSA. The binding stoichiometry is 1:1. The main advantage of this dimeric dye as a non-covalent label is that the free dye has negligible fluorescence.