Simultaneous determination of valsartan, amlodipine besylate and hydrochlorothiazide using capillary zone electrophoresis (CZE).

A capillary zone electrophoresis method was developed for the simultaneous determination of valsartan (VAL), amlodipine besylate (AML) and hydrochlorothiazide (HCZ) in their combined tablets. Separation was achieved on a fused silica capillary by applying a potential of 15 kV (positive polarity) and a running background electrolyte containing 40 mM phosphate buffer at pH 7.5 with UV detection at 230 nm. The samples were injected hydrodynamically for 3s at 0.5 psi and the temperature of the capillary cartridge was kept at 25 degrees C. Pyrazinoic acid was used as an internal standard. The method was validated according to ICH guidelines regarding specificity, linearity, limits of detection and quantitation, accuracy and precision, (Supplementary materials, Table S2). The method showed satisfactory linearity in the ranges of 10-200, 2-20 and 2-20 µg mL(-1) with LODs of 1.82, 0.39, 0.65 µg mL(-1) and LOQs of 5.51, 1.17, 1.96 µg mL(-1) for VAL, AML and HCZ, respectively. The proposed method was successfully applied for the analysis of the studied drugs in their laboratory prepared mixtures and co-formulated tablets. The results were compared with reported methods and no significant differences were found. The proposed method can be used for quality control of the cited drugs in ordinary laboratories.

Micelle-enhanced spectrofluorimetric method for determination of sitagliptin and identification of potential alkaline degradation products using LC-MS.

A novel, quick, simple and highly sensitive spectrofluorimetric method was developed and validated for the determination of sitagliptin (SG) in its pharmaceutical formulations. The proposed method is based on investigation of the fluorescence spectral behavior of sitagliptin in an SDS micellar system. In an aqueous solution of phosphate buffer pH 4.0, the fluorescence intensity of SG in the presence of SDS was greatly enhanced, by 200%, i.e. twofold enhancement. The fluorescence intensity of SG was measured at 300 nm after excitation at 270 nm. The method showed good linearity in the range 0.03-10.0 µg/mL with a good correlation coefficient (r = 0.9998). The limits of detection and quantitation values were 5.31 and 16.1 ng/mL, respectively. The proposed method was successfully applied to the analysis of SG in its single and co-formulated commercial tablets; the results were in good agreement with those obtained using a reference method. Application of the proposed method was extended to stability studies of SG after exposure to different forced degradation conditions according to the ICH guidelines, such as acidic, alkaline, thermal, photo- and oxidative stress. The chemical structure of certain potential degradation products (DPs) were investigated using LC-MS.

Binding constant determination of drugs toward subdomain IIIA of human serum albumin by near-infrared dye-displacement capillary electrophoresis.

Drug binding to serum albumin influences several important pharmacological properties such as toxicity, solubility, activity, distribution, and excretion. It is therefore of interest to have methodologies that allow for the determination of drug-albumin affinity constants while simultaneously providing information on the location of the drug binding site. In the present work we describe a method for the determination of binding constants of drugs known to bind to subdomain IIIA of serum albumin. Drugs used in the study were ketoprofen, ibuprofen, quinidine, naproxen, imipramine, and clofibrate. Binding constants of the drugs were determined by near-infrared dye-displacement capillary electrophoresis. The dye-displacement technique uses a competitive-type interaction between the drug of interest and a dye probe to arrive at a binding constant. A heptamethine cyanine dye was used as a probe for drug binding at subdomain IIIA of serum albumin. The utility of the dye as a noncovalent label for serum albumin was investigated. Additionally, the ability of the method to illustrate enantioselective binding is shown. The dye displacement technique has advantages over current electrophoresis-based techniques in that it is faster and uses less reagent.

Use of non-covalent labeling in illustrating ligand binding to human serum albumin via affinity capillary electrophoresis with near-infrared laser induced fluorescence detection.

This paper demonstrates the use of a near-infrared (NIR) dye as a non-covalent label for human serum albumin (HSA). The dye is a water soluble, heptamethine cyanine dye. The utility of the dye as a tracer illustrating the binding of various drugs to HSA is demonstrated via affinity capillary electrophoresis with near-infrared laser-induced fluorescence detection (ACE-NIR-LIF). Additionally, the factors affecting the separation of relevant species were investigated. The change in quantum yield of the dye upon complexation with HSA was calculated. Spectrophotometric measurements were conducted to study the stoichiometry of the dye albumin complex.

Capillary electrophoresis-based immunoassay for insulin antibodies with near-infrared laser induced fluorescence detection.

A noncompetitive capillary electrophoresis (CE)-based immunoassay with near-infrared laser induced fluorescence detection (NIR-LIF) for insulin antibodies has been developed. In the assay, insulin was derivatized with a NIR fluorescent dye (NN382; LI-COR). Insulin antibodies were detected via the formation of an immunocomplex. Parameters affecting the separation such as pH, voltage and ionic strength were investigated. Furthermore, it was found that increasing the ramp time of the applied voltage improved the detection limit of the assay by an order of magnitude. The detection limit of the assay was 1.1 nM.

Determination of protein-dye association by near infrared fluorescence-detected circular dichroism.

Near-infrared (NIR) squarylium dye spectral properties were evaluated by absorption, fluorescence, circular dichroism (CD), and fluorescence-detected circular dichroism (FDCD). Substituents of the two NN dyes differed at R(1) and R(2), located symmetrically on the chromophore. The side chains of NN525 are R(1)=hexanoic acid, R(2)=butyl sulfonate and R(1)=R(2)=ethyl for NN127. FDCD signals were first confirmed by denaturing BSA with 2-8 M urea showing a diminution of dye FDCD peaks, but no change occurred in spectral properties of the dyes in urea. This indicated that the observed cotton effects occurred by noncovalent interactions with the secondary structure of the protein. The average BSA-dye association constants found by fluorescence, absorbance, and FDCD were 1.27 x 10(6) (n=1) and 3.3 x 10(6) M(-1) (n=1) for NN127 and NN525 respectively. These values were in good agreement when calculated by the three spectroscopic methods validating the use of NIRFDCD for optical parameter calculations. These results are useful to describe NIR squarylium dye labeling of BSA.

Determination of testosterone and 6beta-hydroxytestosterone by gas chromatography-selected ion monitoring-mass spectrometry for the characterization of cytochrome p450 3A activity.

A method for the determination of testosterone and its metabolite, 6beta-hydroxytestosterone, in liver microsomal incubates employing gas chromatography with selected ion monitoring mass spectrometric detection (GC-SIM-MS) has been developed. The method is more rapid than previously reported methods. Testosterone and its metabolites are extracted from the incubation mixture in a single step with methylene chloride. The method does not require derivatization and testosterone and its metabolites are separated on a HP-5MS fused-silica capillary column in less than 15 min. The retention times of testosterone (m/z 288), methyltestosterone (m/z 302), and 6beta-hydroxytestosterone (m/z 304) are approximately 12.7, 12.8, and 13.4 min, respectively. There are no interferences from other known CYP450 metabolites of testosterone. In addition, the selectivity and specificity of the mass spectrometer helps eliminate possible interferences from drugs and new chemical entities evaluated using this methodology. Calibration curves for testosterone and 6beta-hydroxytestosterone are linear from 0.25 to 100 microM. Extraction recoveries are better than 92% for both analytes and the internal standard, methyltestosterone. Over the course of five separate runs, within-day and inter-day precision (expressed as relative standard deviation) was less than 5% for all concentrations of testosterone and 6beta-hydroxytestosterone. Accuracies ranged from 95.8 to 105.8% for testosterone and 94.6 to 104.2% for 6beta-hydroxytestosterone. The assay has been used to characterize the CYP3A metabolic activity of multiple preparations of human, rat, and dog liver microsomes.

Detection of biomolecules in the near-infrared spectral region via a fiber-optic immunosensor.

The design, development, and application of a fluorescent fiber-optic immunosensor (FFOI) procedure for the detection of antibody/antigen binding within the near-infrared (NIR) spectral region is reported. The technique was developed through the combined use of fiber-optics, semiconductor laser excitation, fluorescence detection, NIR dye, and immunochemical techniques. The antibody is immobilized on the FFOI's sensing tip and utilized as a recognition component for trace amounts of specific antigen. The FFOI is constructed to utilize antibody sandwich technique. Three individual immunoassays are reported. The first two assays utilize the FFOI and NN382, a commercial NIR dye, for the detection of human immunoglobulin G (IgG). In these assays, goat anti-human IgG antibody (GAHG) is immobilized on the sensitive terminal of the FFOI followed by the exposure of the antibody-coated terminal to human IgG. The probe is then introduced to GAHG labeled with NN382, generating a signal. The third assay utilizes the FFOI for the detection of trace amounts of Legionella pneumophila serogroup 1 (LPS1). In this assay, rabbit anti-LPS1 antibody is immobilized on the sensitive terminal of the FFOI followed by exposure to LPS1. The antigen-coated probe is then treated with monoclonal anti-LPS1 antibody followed by incubation with GAHG labeled with NN382. The assays are optimized to detect the corresponding antigen via the NIR-FFOI. Typical measurements are performed in 10-15 min. A 780-nm semiconductor laser provides the excitation of the immune complex and the resulting emission is detected by a 820-nm silicon photodiode detector. The intensity of the resulting fluorescence is directly proportional to the concentration of the antigen. Solutions of IgG and LPS1 with concentrations as low as 10(-11) M and 0.5 ng/ml, respectively, have been detected with a minimum interference.

Ultrasensitive detection of closely related angiotensin I peptides using capillary electrophoresis with near-infrared laser-induced fluorescence detection.

A novel near-infrared (NIR) fluorescent dye (NN382, LICOR, Inc.) was evaluated as an ultrasensitive peptide-labeling reagent for use with capillary electrophoresis (CE). Six angiotensin I (Ang-I) variants were selected as model peptides for the derivatization and separation studies. The closely related decapeptides were labeled with the NIR dye, separated using CE, and detected by NIR laser-induced fluorescence. Derivatization of the peptides was achieved under aqueous conditions using 2.5-500 pmol of Ang-I in a 50-microL sample (5 x 10(-8)-1 x 10(-5)M), and between 1.3 and 254 amol of the labeled peptides were injected on column. The fluorescence response was linear over a 200-fold range (correlation r > or = 0.9986). The limit of detection (SNR = 3, signal/RMS noise) ranged from 100 to 300 zmol, for the six Ang-I variants. Four of six peptides were resolved from each other and excess dye using capillary zone electrophoresis with a simple 50 mM phosphate run buffer, pH 7.2. Two pairs of coeluting peptides were successfully resolved using micellar electrokinetic chromatography with a nonionic surfactant, Triton X-100. The NIR amine-labeling reagent NN382 is a viable alternative to using visible fluorophores for CE methods requiring high sensitivity.

Effects of organic and aqueous solvents on the electronic absorption and fluorescence of chloroaluminum (III) tetrasulphonated naphthalocyanine.

The effects of various solvents on the ground and excited states of chloroaluminum (III) tetrasulphonated naphthalocyanine (AlNcS(4)) were studied. Both the absorbance and fluorescence spectra were found to be influenced by the hydrogen bond donating ability of various solvents. As the hydrogen bond donating ability of the solvent increased, hypsochromic and bathochromic shifts in the absorbance and fluorescence spectra were observed in protic and aprotic solvents respectively. Plots of the absorbance and fluorescence maxima versus the E(T)(30) solvent parameter showed linear relationships in binary mixtures of protic-protic (methanol-H(2)O) and aprotic-protic (DMSO-H(2)O) solvents. Aggregation was indicated by a broad band in the ground state absorption spectra and a low quantum efficiency 0.04 relative to the efficiency observed in organic solvents. A face-to-face conformation of the monomeric subunits of the dimer is suggested due to the red-shifted absorbance band. The acid-base properties of the dye were studied and were indicative of a multi-step process. In acidic conditions (pH 1), protonation of the bridging nitrogen atoms was identified by a broad band appearing red-shifted to those obtained at higher pH values. Under slightly acidic conditions a pKa value of 6.7 was determined for one of the meso-nitrogen. In alkaline conditions a pKa of 11.5 was determined for another meso-nitrogen and a second fluorescence band emerged at 804 nm, red-shifted to the emission maxima.

Spectral characterization of a novel near-infrared cyanine dye: a study of its complexation with metal ions.

The spectral features of the near-infrared (NIR) dye TG-170 in different solutions and its complexation with several metal ions were investigated. The absorbance maxima of the dye are at lambda=819, 805, and 791 nm in dimethyl sulfoxide (DMSO), methanol, and a buffer of pH 5.9, respectively. These values match the output of a commercially available laser diode (780 nm), thus making use of such a source practical for excitation. The emission wavelengths of the dye are at lambda(em) =822, 812, and 803 nm in DMSO, methanol, and the buffer, respectively. The molar absorptivity and fluorescence quantum yield increase accordingly. The addition of either an Al(III) ion or Be(II) ion resulted in fluorescence quenching of the dye. The Stern-Volmer quenching constant, K(SV), was calculated from the Stern-Volmer plot to be K(SV)=3.11x10(5) M(-1) for the Al(III) ion and K(SV)=1.17x10(6) M(-1) for the Be(II) ion. The molar ratio of the metal to the dye was established to be 1:1 for both metal ions. The stability constant, K(S), of the metal-dye complex was calculated to be 4.37x10(4) M(-1) for the Al-dye complex and 1.94x10(6) M(-1) for the Be-dye complex.

Fluorescence studies of carbocyanines using AOTF.

Over the past few years acceptance of acousto-optic devices which can control optical radiation has increased. In this paper the use an acousto-optic tunable filter (AOTF) within an existing spectrofluorometer as a replacement for the excitation monochromator is reported. Long wavelength absorbing carbocyanine dyes that have strong absorption and high quantum yield are used as standards. The major advantage of using an AOTF filter is the wavelength purity and that it can act as a wavelength selector in place of a monochromator. The use of AOTF in place of bandpass filters for removing laser diode side bands is also discussed.

Near-infrared tetra-substituted aluminum 2, 3-naphthalocyanine dyes for optical fiber applications.

The synthesis and spectral characterization of several tetra-substituted aluminum 2, 3-naphthalocyanine dyes for the determination of metal ions is reported. The synthesis is done by means of a homogeneous phase reaction, replacing the previously used heterogeneous method. The new scheme allows for improved product yields, higher purity, better product reproducibility and can be monitored at different stages using UV-Vis-near-infrared spectroscopy. The incorporation of electron-donating or -withdrawing groups was found to influence the product yield and to cause a shift in the absorbance maximum. The typical shift in the excitation maximum (of up to 27 nm) enables the dye to match the output of semiconductor laser diodes. In addition the tetra-substituted groups were capable of undergoing an ion-exchange process with the metal ions which produced a change in the fluorescence signal of the dye. Similar results were achieved using an optical fiber metal probe. The detection of metal ions using the near-infrared dyes was accomplished via steady-state fluorescence using both a commerically available instrument and a fiber optic system and also via the fluorescence lifetime technique.

Design and development of a fiber-optic immunosensor utilizing near-infrared fluorophores.

The design and application of a fluorescent fiber-optic immunosensor (FFOI) are reported. The FFOI is utilized for the detection of antibody/antigen binding within the near-infrared (NIR) spectral region. The technique is developed through the combined use of fiber-optic, semiconductor laser-excitation, fluorescence detection, NIR dye, and immunochemical techniques. The antibody is immobilized on the FFOI and utilized as a recognition component for trace amounts of specific antigen. The FFOI is constructed to utilize an antibody sandwich technique. The assay involves the immobilization of the capture antibody on the sensing tip of the FFOI followed by the exposure of the immobilized sensing tip to the antigen. The antigen-coated FFOI is then introduced to a second antibody previously labeled with the NIR dye. Typical measurements are performed in about 15 min. A semiconductor laser provides the excitation (780 nm) of the immune complex. The resulting emission is detected by a silicon photodiode detector (820 nm). The intensity of the resulting fluorescence is directly proportional to the concentration of the antigen. The sensitivity of the analysis reaches 10 ng/ml and the response time is 10-15 min.

Direct determination of substituted azepinoindole enantiomers in rat plasma using silica stationary phase and beta-cyclodextrin as a mobile phase additive.

DU 124884 is a racemic serotonin receptor agonist in an early stage of drug development. DU 124884 and its potential N-desmethyl metabolite, KC 9048, both contain a single chiral center. A direct enantioselective HPLC assay was developed and validated to quantify DU 124884 and KC 9048 in rat plasma. The drug and metabolite enantiomers were extracted from plasma and separated using silica stationary phase with an aqueous mobile phase containing beta-cyclodextrin (beta-CD), triethylamine, and 2-methyl-2-propanol. A variable wavelength detector was used to monitor absorbance at 231 nm. The assay calibration range was from 100 to 5000 ng/mL. Quality control sample precision (< or = 9% RSD) and accuracy (+/-10% error) were satisfactory for all four analytes (n = 12). The method was used to assess drug exposure during a pilot toxicology study in rats. Toxicokinetic study animals were dosed subcutaneously for 15 days at 0, 2.5, 10, and 40 mg of DU 124884.HCl kg-1 day-1. Blood was collected on the last day of dosing between 22 min and 4 h and 13 min after the last dose. The samples showed (+/-)-DU 124884 isomer ratios ranging from 1.1 to 1.3. These data suggest that DU 124884 undergoes stereoselective metabolism in rats. Levels of the N-desmethyl metabolite enantiomers were < 100 ng/mL.

Instrument to detect near-infrared fluorescence in solid-phase immunoassay.

The construction of a near-infrared (near-IR) fluorescence detector for measuring picomolar levels of near-IR laser dyes is described. The detector is designed for use in an immunoassay technique that employs antibodies labeled with near-IR polymethine cyanine dyes. These dyes possess spectral properties that are exclusive to the near-IR region (650-1100 nm). The instrumentation is characterized, including its hardware and data acquisition software components. The detector is capable of measuring fluorescence in both solution and solid-phase environments. Data on the detector's performance is presented.

Spectroscopic studies of a near-infrared absorbing pH sensitive aminodienone-carbocyanine dye system.

Synthetic red and near-infrared absorbing dyes may be used as probe molecules in a large number of applications. Dyes exhibiting spectral changes with hydrogen ion concentration are useful as pH probes. Those dyes which have their absorption and fluorescence maxima in the long wavelength region of the visible spectral region are specially valuable because of decreased interference and semiconductor laser applications. In this paper we have evaluated an aminodienone dyes 1 which demostrates pH dependent absorption and fluorescence spectra as well as solvent polarity dependence. In organic solvents the long wavelength absorption band of the dye is in the reduced interference region. The absorption maximum is at 535 nm in neutral or alkaline solutions in methanol. The absorption spectra undergo a strong bathochromic shift in the presence of acids (lambda(max) = 709 nm) with a concomitant change in the fluorescence spectra. This pH sensitive dye was found to be specially especially useful for organic solvents. The analytical utility of this and similar near-infrared absorbing dyes is discussed.

Comparison of covalent and noncovalent labeling with near-infrared dyes for the high-performance liquid chromatographic determination of human serum albumin.

Noncovalent and covalent methods of labeling protein with near-infrared polymethine cyanine dyes were compared for use in analyzing human serum albumin (HSA) by high-performance liquid chromatography (HPLC) with near-infrared absorbance detection. While noncovalent labeling was faster than covalent labeling and took place in the physiological pH range, covalent labeling was more stable under conditions encountered in many of the widely used types of HPLC. Covalently labeled HSA protein peaks indicated uniform labeling of amino groups at both hydrophilic and hydrophobic binding sites, while noncovalent labeling showed a preference for hydrophobic binding sites.

Spectroscopic studies of a near-infrared absorbing pH sensitive carbocyanine dye.

Near-infrared dyes can be derivatized with appropriate functional groups for use as analytical probes for numerous applications. A dye derivatized with pH-sensitive functional groups may show spectral changes when the pH of its environment is changed. These dyes are valuable to the researcher since they absorb and fluoresce at long wavelengths where interference is minimal and their absorption maxima permit the use of semiconductor lasers. In this paper we have evaluated the sensitivity to pH of a bis-carboxylic acid derivative of a near infrared dye to illustrate its potential as a probe for determining pH. The dye has an absorption maximum at 795 nm in aqueous solution, a region where several commercially available laser diodes have their output maximum. The analytical utility of near-infrared laser diodes for pH determination has also been evaluated.

The utility of time-resolved emission spectroscopy in the study of cyclodextrin-pyrene inclusion complexes.

The application of time-resolved emission spectroscopy to the characterization of cyclodextrin inclusion complexes of pyrene in the presence of various alcohols is described. Such measurements offer a means of selectively studying the characteristics of inclusion complexes in the presence of uncomplexed pyrene. The fluorescence lifetimes and formation constants of these complexes are enhanced in the presence of alcohols. These enhancements are reportedly due to the formation of pyrene-alcohol-CD ternary complexes.