Ultraviolet Densitometry Research Articles

Development and validation of a simple and rapid thin-layer chromatography–UV densitometry method for the determination of triazophos in human whole blood for forensic toxicological applications

No published studies reported triazophos (TZP) analysis in human whole blood following fatal intoxication. In this study, a method for the determination of TZP in human whole blood using liquid–liquid extraction (LLE) and thin-layer chromatography (TLC)–ultraviolet (UV) densitometry is described. Chromatography was performed on silica gel 60F254 TLC plates using mobile phase n-hexane:acetone (8:2, v/v) and UV densitometric detection at 248 nm. Better extractions were achieved by using ethyl acetate solvent at pH 5 with a recovery of 92.17%. Calibration curve for TZP in blood was linear from 2 to 100 μg/mL (0.04 to 2 μg/spot) and the sensitivity of the method (LLOQ) was 2 μg/mL (0.04 μg/spot). The method showed excellent within-day precision (0.37 to 0.82%, RSD) and between-day precision (0.39 to 1.47%, RSD) for spiked blood samples at 2, 10, and 50 μg/mL. The measured concentrations of TZP in blood did not deviate more than 2.69% under different storage conditions. TZP concentrations in two fatal cases of poisoning were reported. To the best of our knowledge, this is the first validated TLC–UV densitometric method developed to determine TZP in human whole blood.

Simultaneous quantitation of nicotinamide riboside and nicotinamide in dietary supplements via HPTLC–UV with confirmation by online HPTLC–ESI–MS

The identification and quantitation of nicotinamide riboside (NAR) and its main related compound (nicotinamide) were achieved using high-performance thin-layer chromatography (HPTLC)–ultraviolet (UV) densitometry with confirmation by online electrospray ionization (ESI)–mass spectrometry (MS). As the stationary phase, HPTLC Si 60 F254 glass plates were employed; the mobile phase was ethanol–1 M ammonium acetate–formic acid (7:1:0.1, v/v/v). No derivatization was applied, and UV densitometry was performed in the absorbance mode (270 nm). The method was validated by specificity, linearity, accuracy, precision, and robustness.

Determination of Oxazepam in Pharmaceutical Formulation by HPTLC UV-Densitometric and UV-Derivative Spectrophotometry Methods

Methods for determination of oxazepam in pharmaceutical formulation by derivative ultraviolet (UV) spectrophotometry as well as high-performance thin-layer chromatography (HPTLC) UV densitometry were described. For UV-derivative spectrophotometry, some derivatives and wavelengths may be recommended for routine quality control of the drug of interest. On the other hand, HPTLC provided good results, but only when the calibration curve was estimated using nonlinear regression analysis. The HPTLC method was developed with silica F254 plates, a mobile phase of benzene/ethanol (5:1, v/v), and densitometric detection at 204 nm receiving R f = 0.47. Developed methods were validated and found to be sufficiently precise and reproducible for established conditions.

Analysis of IgA1 O-glycans in IgA nephropathy by fluorophore-assisted carbohydrate electrophoresis.

Abnormal O-glycosylation of IgA1 may contribute to pathogenic mechanisms in IgA nephropathy (IgAN). Observations of altered lectin binding to IgA1 in IgAN suggest that the O-glycan chains may be undergalactosylated, but precise structural definition of the defect has proved technically difficult, and it remains unconfirmed. This is the first study using fluorophore-assisted carbohydrate electrophoresis (FACE) to analyze IgA1 O-glycans in IgAN and controls. IgA1 was purified from serum, and the intact O-glycans were released by hydrazinolysis at 60 degrees C. After re-N-acetylation, the glycans were fluorophore-labeled and separated by polyacrylamide gel electrophoresis. Sequential exoglycosidase digestions of IgA1 allowed identification of the different O-glycan bands on FACE gels, and their relative frequencies in IgA1 samples were measured by ultraviolet densitometry. Lectin binding of the IgA1 samples was also measured. In some patients with IgAN, FACE analysis demonstrated a significant increase in the percentage of IgA1 O-glycan chains consisting of single N-acetyl galactosamine (GalNAc) units rather than the more usual galactosylated and sialylated forms. This finding was confirmed using both desialylated IgA1 and enzymatically released O-glycans. Good correlation was also found between O-glycan agalactosylation on FACE analysis and IgA1 lectin binding in IgAN, supporting the value of lectins as tools for detection of this abnormality. This is the first study in which all of the predicted O-glycan forms of IgA1 have been analyzed simultaneously, and demonstrates that in IgAN, the IgA1 Oglycan chains are truncated, with increased terminal GalNAc. This abnormality has the potential to significantly affect IgA1 behavior and handling with pathogenic consequences in IgAN.

Simplified method for simultaneous determination of diazepam and its metabolites in urine by thin-layer chromatography and direct densitometry

A direct densitometric method for determination of diazepam and its metabolites in urine was developed. The proposed procedure involves acid hydrolysis of urine specimens, thereby converting diazepam and its metabolites into benzophenones [2-methylamino-5-chlorobenzophenone (MACB) and 2-amino-5-chlorobenzophenone (ACB)]. It is followed by extraction with chloroform—isopropanol (3:1, v/v). The two benzophenones were separated on thin-layer chromatography plates using hexane—diethyl ether—acetic acid (80:10:10) as a mobile phase. Quantitation of the MACB and ACB spots was achieved by direct ultraviolet densitometry. The limit of detection was 0.5 μg per ml of urine for both benzophenones. The proposed method is simple, rapid, reproducible and has been found to be effective for direct determination of diazepam and its metabolites in urine.

Determination of nitrazepam and its main metabolites in urine by thin-layer chromatography and direct densitometry

A method for the direct quantitative densitometry of nitrazepam and its main metabolites (7-aminonitrazepam, 7-acetamidonitrazepam and 2-amino-5-nitrobenzophenone) in urine was developed. The unchanged drug and its metabolites were extracted with benzene—dichloromethane (4:1), subjected to thin-layer chromatography, and determined by direct ultraviolet densitometry. Recovery experiments showed that the method was quantitative. The limit of detection was 5 ng/ml for 2-amino-5-nitrobenzophenone and 10 ng/ml for other compounds. The method was applied to the determination of nitrazepam and its metabolites excreted in human urine after administration of 10 mg of the drug.

Analysis of Pharmaceuticals by Ultraviolet Densitometry on Thin-Layer Chromatograms I: Parabens in Gels and Creams

Precision of about 1 percent and excellent recovery are attainable by quantitative densitometry of ultraviolet chromophores separated by thin-layer chromatography on plates containing a phosphor. Quenching of the fluorescent background affords a sensitive measure of concentration. Substrates with native or derived fluorescence can be determined on plates by fluorometry using either a nonfluorescent background or a phosphor that fluoresces at different wavelengths. This report describes application of these and similar techniques to the determination of parabens in pharmaceutical and cosmetic products. The densitometry technique requires simple solvent extraction of the paraben-preserved product and chromatography on silica gel thin layers containing phosphor. Use of a 4:1 pentane-acetic acid system provided resolution of the parabens from each other, and no interference was encountered with hydrocortisone acetate, partial sodium salt of n-lauryl β-imino diproprionic acid, EDTA, polyethylene glycol, hydroxyethyl cellulose, hydroxypropyl methylcellulose, D & C Green No. 5 dye, and various waxes and fats. Preliminary investigations for applying direct ultraviolet densitometry to other types of thin layers are mentioned.

Direct spectrophotometric analysis of ribonucleic acid fractionation by agar-gel electrophoresis

The fractionation of rat-liver RNA was studied by direct ultraviolet densitometry of dry agar electrophoregrams. Highly reproducible electrophoretic patterns of phenol extracted RNA and residual RNA were obtained. Phenol-extracted RNA was resolved into 3 main and 4–6 minor fractions. The main fractions accounted in most cases for 90 % of the absorbancy area and corresponded to the two components of ribosomal RNA and soluble RNA. Residual RNA extracted with phenol at 65° in the presence of sodium dodecylsulphate was resolved into 11–13 fractions, 3 of them being components of ribosomal RNA and low-molecular-weight RNA. A slow-moving region was also present in which the two slowest components are extracted only in the presence of sodium dodecylsulphate.

The Estimation of Serum Proteins by Ultraviolet Densitometry of Agar Electrophoretic Diagram

The Estimation of Serum Proteins by Ultraviolet Densitometry of Agar Electrophoretic Diagram