The development of metalloporphyrin‐ (ferric protoporphyrin IX chloride (FePP), cobalt (III) protoporphyrin IX chloride, copper (II) protoporphyrin IX) enhanced chemiluminescent (CL) imaging ...detection of serum proteins after PAGE is described in this article. The detection is based on the catalytic activity of metalloporphyrins, especially FePP, in the CL reaction of the luminol–H2O2 system. Some relatively low abundant proteins such as hemopexin (Hpx) and complement C4 are sensitively detected by FePP‐enhanced CL imaging. Other proteins such as haptoglobin, apolipoprotein A‐1, complement C3, and α‐1‐antitrypsin are also detected and identified by MS and MS/MS techniques. Detection limit of Hpx is as low as 20 ng, without the need of expensive antibodies or tedious immunoassay procedures. The mechanism of the proposed method is discussed employing standard proteins. The application to the analysis of different protein patterns in healthy people and in Thalassemia patients is being investigated.
A second order polynomial calibration model was developed and statistically validated for the direct and non-destructive quantitative analysis – without sample preparation – of the active ...pharmaceutical ingredient (API) salicylic acid in a pharmaceutical ointment using FT-Raman spectroscopy. The calibration curve was modeled by plotting the peak intensity of the vector normalized spectral band between 757 and 784
cm
−1 against the known salicylic acid concentrations in standards. At this band, no spectral interferences from the ointment vehiculum (white vaseline) are observed. For the validation of the polynomial model, its fit and its predictive properties were evaluated. The validated model was used for the quantification of 25 ointments, compounded by different retail pharmacists. The same standards and samples were used, both for development and validation of a regression model and for quantitative determination by HPLC – with sample preparation – as described for the related substances of salicylic acid in the
Ph. Eur. IV. The quantification results obtained by the FT-Raman method corresponded with the HPLC results (
p
=
0.22), provided that the particle size of salicylic acid in the standards is the same as in the analyzed samples. The non-destructive FT-Raman method is a reliable alternative for the destructive HPLC method, as it is faster and does not require sample pre-treatment procedures.
We provide an overview of powerful applications of chemiluminescence (CL) analysis for biomolecules, mainly in gel electrophoresis. In routine immunoassays, CL labels and detection reactions are ...widely used for peroxidase and alkaline-phosphatase enzymes. The sensitivity, the dynamic range and the diversity of CL assays have led to a vast range of applications, notably in protein and nucleic-acid blotting. Non-enzymatic CL detection is also being developed gradually.
Direct CL detection of biomolecules in gels has emerged recently, with simple, convenient and rapid methods. It offers substantial potential to detect many proteins or nucleic acids in complex biological samples. In addition, metallic nanoparticles and catalytic nucleic acids are also potential candidates for CL detection of biomolecules in the future.
A simple linear regression method was developed and statistically validated for the direct and non-destructive quantitative analysis—without sample preparation—of the active pharmaceutical ingredient ...(API) medroxyprogesterone acetate (MPA) in an aqueous pharmaceutical suspension (150
mg in 1.0
ml) using FT-Raman spectroscopy. The linear regression was modelled by plotting the highest peak intensity of the vector normalized spectral band between 1630 and 1590
cm
−1 against different MPA standard suspension concentrations. At this band, no spectral interferences from additives in the suspension are observed. The validated model was used for the quantification of a commercial suspension (150
mg in 1.0
ml) of the commercialized preparations. The same standards and samples were used, respectively, for the development and validation of a simple linear regression model and for the quantitative determination by means of HPLC—with sample preparation—as described for the related substances of MPA in the
Ph. Eur. IV. The quantification results obtained by the FT-Raman method corresponded with the claimed label concentration (150.01
±
0.96
mg/ml (
n
=
6)). Applying the HPLC method, however, a systematic error was observed (157.77
±
0.94
mg/ml (
n
=
6)). The direct FT-Raman method hence appears the most reliable for the quantification of the MPA component in suspension, compared to the HPLC method that requires sample preparation. The latter method provides a systematic error because the exact volume or density of a suspension sample is unknown. A precise isolation of fixed volumes from a suspension is rather unfeasible because of the continuous sagging of the suspended particles and their sticking to the used materials in the isolation process.
Chemiluminescence, when combined with capillary electrophoresis (CE), offers excellent analytical sensitivity and selectivity. Campana et al summarize progress made in chemiluminescence detection ...for CE and review the applicability of current methodology.
A flow injection analysis method is proposed for the determination of tiopronin based upon the oxidation by cerium(IV) in dilute sulfuric acid medium and sensitized by quinine. With the peak height ...as a quantitative parameter applying optimum working conditions, tiopronin is determined over the 1-400 microM range (150 microliters per injection, n = 10, r = 0.9994) with a detection limit of 0.34 microM and an RSD (n = 10) less than 2% at 20 and 50 microM. The proposed method, combining the advantages of speed and sensitivity, was applied to the routine determination of tiopronin in a pharmaceutical preparation.
The determination of the alkaloids reserpine, rescinnamine and yohimbine based on a chemiluminogenic reaction with potassium permanganate in the presence of polyphosphoric acid is described. The ...investigation was carried out using a batch and a flow injection chemiluminometer. Both approaches were accurate and precise, allowing the measurement of reserpine within the ranges 0.100-3.00 and 0.050-3.00 micrograms ml-1 with RSD values for 1.00 microgram ml-1 of 1.91 and 0.33% (n = 8) with the batch and the flow injection manifold, respectively. The procedure was successfully applied to formulations after extraction of reserpine with chloroform, with recoveries from commercial formulations within the range 95.2-99.0%.
The application of carbon nanotube polyacrylamide gel electrophoresis (PAGE) is being reported so as to improve selectivity of analysis. A novel polyacrylamide gel was manufactured by adding ...multi-walled carbon nanotubes (MWNT) when producing the polyacrylamide gel. Carbon nanotube-matrix assistance was used to separate apolipoprotein A-I and complement C3 with native polyacrylamide gel electrophoresis. Significant improvement of the relative selectivity was achieved in the separation between apolipoprotein A-I and complement C3, because of the hydrophobic change of the carbon nanotube surface non-covalently functionalized by the surfactant Triton X-100. These two proteins were identified by MS/MS after electrophoresis. The change of the observed mobility can also be noticed using other nanomaterials such as nano-TiO
2 and nano-Al
2O
3, but no significant improvement of relative selectivities could be observed with the latter nanoparticles.
