Maria Grazia Gioia

HPLC-fluorescence determination of amino acids in pharmaceuticals after pre-column derivatization with phanquinone

4,7-Phenanthroline-5,6-dione (phanquinone) was used as a fluorogenic labeling reagent in pre-column derivatization for the quality control of amino acids in pharmaceuticals. The amino acid adducts were efficiently separated by C12 RP high-performance liquid chromatography (HPLC) using a ternary mixture of triethylamine (TEA) phosphate buffer (pH 2.5, 0.05 M)-methanol- tetrahydrofuran (THF) as mobile phase by varying composition gradient elution and detected fluorometrically. The results obtained by the proposed method were compared statistically, by means of the Students t-test and the variance ratio F-test, with those obtained by a rapid reference method, which involved o-phthaldialdehyde (OPA) as pre-column reagent; no significant difference was found. The stronger derivatization conditions (60 degrees C, pH 8, 60 min) required for the method with phanquinone are compensated by the major stability of derivatives and by the absence of fluorescent degradation products.

Serum Thyroid-Stimulating Hormone as a Predictor of Cognitive Impairment in an Elderly Cohort

Background: It is unclear whether in late life serum thyroid-stimulating hormone (TSH) predicts risk of developing cognitive impairment. Objective: This study investigated the prospective relationship of serum TSH with the risk of developing mild cognitive impairment (MCI), Alzheimer’s disease (AD) and vascular dementia (VaD) in an elderly cohort with a 4-year follow-up. Methods: Data are for 660 subjects aged 65 years and older from an Italian population-based cohort who were cognitively normal at an extensive assessment in 1999/2000 and underwent follow-up assessment in 2003/2004. Serum TSH was measured at baseline. Multinomial logistic models adjusted for sociodemographic and cardiovascular risk factors were used to investigate the association of serum TSH (both as a tertile and continuous log-transformed variable) with risk of incident MCI, AD and VaD diagnosed according to international criteria. Results: Over 3.8 ± 0.7 years of follow-up, there were 149 incident MCI cases (77 with impairment of memory and 72 with impairment of nonmemory domains) and 86 incident dementia cases (53 with AD, 28 with VaD). No association between baseline TSH and risk of developing any MCI subtype or AD was found. The highest TSH tertile had a threefold higher increased risk of VaD (OR: 3.25, 95% CI: 1.01–10.77, p = 0.048) compared to the lowest tertile. Risk of VaD increased about 60% for each 1 SD increase in log-transformed TSH (OR: 1.61, 95% CI: 1.06–2.44, p = 0.025). Conclusions: In this elderly cohort, baseline TSH was not related to the risk of developing MCI or AD, but high TSH was associated with an increased risk of VaD. These results suggest further need for research using larger samples to examine the role of TSH as a predictor of VaD and the role of thyroid autoimmunity in vascular cognitive impairment.

2,5-Dimethyl-1H-pyrrole-3,4-dicarbaldehyde as a precolumn derivatization reagent for HPLC/UV detection of amino acids

The use of 2,5-dimethyl-1H-pyrrole-3,4-dicarbaldehyde as a precolumn derivatization reagent for HPLC analysis of amino acids is proposed. The compound reacts under mild conditions (10min at ambient temperature) with primary amino groups. The derivatization conditions to obtain quantitative reaction were optimised by considering different parameters (temperature, pH and reagent concentration) using l-Val as the model compound. The synthesized l-Val derivative was characterized by (1)H NMR and UV. The derivatives of 19 amino acids were separated by reversed-phase HPLC and detected at lambda=320nm. The method was applied successfully to the qualitative and quantitative analysis of commercial polyamino acid preparations.

Determination of retinoids in galenicals by column liquid chromatography with fluorescence and diode-array detection

Simple and rapid reversed-phase gradient column liquid chromatography (LC) with fluorescence detection at different wavelengths was developed for the simultaneous analysis of all-trans, 13-cis, 9-cis retinoic acids, vitamin A palmitate and beta-carotene in galenicals. The assay results agreed with those obtained by an LC method with diode-array UV detection. A post-column on-line photochemical reactor (irradiation at 254 and 366 nm) was inserted between the LC column and the fluorescence detector to enhance the performance of the method. Two fluorescence spectra (photoreactor on and off) were obtained for each analyte which proved useful for the unambiguous identification of the various analytes.

HPLC-fluorescence determination of unconjugated estrogens in pharmaceuticals.

A fluorimetric liquid chromatographic method (lambda(ex) = 280 nm; lambda(em) = 312 nm) was developed for measurements of unconjugated estrogens (estradiol and estriol) in pharmaceutical dosage forms using a reversed-phase column with water acetonitrile at different composition as mobile phase. The in vitro release profiles of three different estradiol transdermal therapeutic systems were determined through a medical-grade silicone rubber subdermal implant material membrane, using a modified Franz diffusion apparatus at 37 degrees C in presence of PEG 400. The HPLC method possesses advantages of rapidity, simplicity and accuracy.

HPLC analysis of pharmaceutical estrogens in raw materials and dosage forms

The use of HPLC with fluorescence detection (lambda ex = 280 nm; lambda em = 410 or 312 nm) in combination with a postcolumn on line photochemical derivatization was investigated for the analysis of conjugated and unconjugated estrogens and their correlated impurities. The column effluents were subjected on-line to UV irradiation (254 nm) and the photo induced modifications were useful for the identification of the various estrogens. The proposed HPLC methods were successfully applied to the analysis of commercially available conjugated estrogens (raw materials and pharmaceuticals) and estrogen samples. The assays results relative to the pharmaceutical formulations were in agreement with those obtained by a reference HPLC method with UV detection (lambda = 280 nm).

4,7-Phenanthroline-5,6-dione: A New Labelling Reagent for Liquid Chromatographic Analysis of Amino Acids

SummaryThe use of 4,7-phenanthroline-5,6-dione (phanquinone) as a pre-chromatographic reagent for LC analysis of amino acids is proposed. The reagent reacts (30 min at 50°C) with primary amino group and the resulting adducts can be chromatographed under reversed-phase conditions (Phenyl-Hexyl column). The experimental conditions for the derivatization and chromatographic separations are discussed. Application to the determination of several amino acids in pharmaceutical formulations is described.

HPLC-fluorescence determination of equilin and equilenin in postmenopausal women's urine.

A high-performance liquid chromatographic (HPLC) method with fluorescence detection (lambda(ex) = 280 nm; lambda(em) = 410 and 312 nm) in combination with a post-column on-line photochemical derivatization is described for the determination of equilin and equilenin in urine from normal postmenopausal women after therapy with conjugated oestrogens. The column effluents were subjected on-line to UV irradiation (254 nm) and the photo-induced modifications were useful for the identification of the analytes. The conjugated (sulphate and glucuronide) forms were analysed after enzymatic or chemical hydrolysis and extracted with chloroform. Solid-phase extraction using strong anion-exchange sorbent was applied to the analysis of unconjugated oestrogen fraction to obtain a practical and reliable sample clean-up. The HPLC separations were achieved using ODS columns with a mobile phase consisting of 0.05 M triethylamine phosphate buffer (pH 4.0)-acetonitrile (64:36, v/v) at a flow rate of 1.0 mL/min. The method was accurate and reproducible; for the equilin and equilenin separation isocratic conditions were satisfactory, allowing a sensitive detection in urine samples with a detection limit of about 50 fmol for equilin (lambda(ex) = 280 nm; lambda(em) = 312 nm, after photoderivatization) and 10 fmol for equilenin (lambda(ex) = 280 nm; lambda(em) = 410 nm).

Validation of a spectrophotometric method for the determination of iron (III) impurities in dosage forms

A spectrophotometric method (lambda=535 nm) for the iron (III) impurities determination in iron protein-succinylate complex syrup using thioglycolic acid in basic ambient was proposed and validated. Assay samples were treated with 0.1 N hydrochloric acid and centrifuged to remove the interfering active drug. Linear response (r=0.9999) was observed over the range of 0.005-0.2% of the iron (III) with respect to the complex nominal concentration. The accuracy could be considered very satisfactory (recovery=97-99%). The intra-day precision (RSD) of impurity amongst six independent sample preparations, was 1.4%, and there was no significant difference between intra- and inter-day studies. Intermediate precision indicated that the assay possessed high degrees of ruggedness. The limit of quantitation was 0.005% of impurity with respect to the active drug. The results obtained for iron (III) were compared statistically with those obtained with the standard addition method by means of the Students t-test and the variance ratio F-test; no significative difference was found.

A SIMPLE AND VALIDATED LC METHOD FOR THE SIMULTANEOUS ANALYSIS OF GLUCOSAMINE AND CHONDROITIN SULFATE EQUIVALENT IN DIETARY PRODUCTS

A simple and reliable reversed-phase liquid chromatographic (RP-LC) method was developed and validated to determine simultaneously a glucosamine and chondroitin sulfate equivalent in dietary products. The procedure is based upon the reaction of o-phthaldialdehyde with glucosamine and galactosamine coming from the galactosaminoglycan hydrolysis. The hydrolysis reaction was carried out with hydrochloric acid (7.5 N) at 80°C for 8 hr, whereas, the pre-column derivatization reaction was carried out in alkaline media for 1 min at ambient temperature. The chromatographic separations were performed on a Phenomenex Synergi 4 μ fusion-RP 80 A (250 mm × 3.0 mm i.d.) using a mobile phase consisting of a mixture of sodium acetate buffer (pH 5.9; 0.05 M) and methanol (85:15, v/v). UV-DAD detection at λ = 340 nm was used. Linear responses were observed and the limit of quantitation for both aminosaccharides was about 60 pmol. The intra-day precision (RSD) was ≤1.8% and there was no significant difference between intra- and inter-day data. Recovery studies showed good results (99.3–101.0%) with RSD ranging from 1.1 to 2.1%.