Rita Gatti

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.

Analysis of pharmaceutical creams: a useful approach based on solid-phase extraction (SPE) and UV spectrophotometry

Solid-phase extraction (SPE) using C-18, diol and ion-exchange sorbents followed by UV spectrophotometric (conventional and derivative mode) assay was applied to the analysis of basic, acidic and neutral drugs commercially available in creams. A representative set of drugs (promethazine, chlorhexidine, benzydamine, ketoprofen, ibuprofen, fentiazac, piroxicam, fluorouracil, crotamiton and hydrocortisone acetate) was selected, and for each drug the appropriate SPE conditions (adsorption, washing and elution) were investigated to obtain a practical and reliable sample clean-up. It was shown that the developed SPE procedures were capable of removing interfering cream components (excipients including preservatives) allowing accurate spectrophotometric analyses to be performed. In some applications, derivative spectrophotometry was advantageous over the conventional absorption mode with respect to higher selectivity and versatility.

High-performance liquid chromatographic determination of aliphatic thiols with aroylacrylic acids as fluorogenic percolumn derivatization reagents

Abstract The use of the methyl ester of 4-(6-methoxynaphthalen-2-yl)-4-oxo-2-butenoic acid as a fluorogenic labelling regent for the high-performance liquid chromatography (HPLC) of biologically important thiols (glutathione, cysteine, acetylcysteine, homocysteine, cysteamine, sodium 2-mercaptoethanesulphonate and thiola) was investigated. The compound reacts selectively and rapidly (10 min at ambient temperature and pH 7.5) with the thiols to give fluorescent adducts that can be separated by reversed-phase HPLC and detected fluororimetrically (λem = 450 nm; λem = 310 nm). Applications to the determination of l -cysteine and mesna in pharmaceutical formulations are described.

1,1′-[Ethenylidenebis(sulfonyl)]bis-benzene: A useful pre-chromatographic derivatization reagent for HPLC analysis of thiol drugs

SummaryThe use of 1,1′-[ethenylidenebis(sulfonyl)]bis-benzene as a pre-chromatographic reagent for LC analysis of thiol drugs is proposed. The reagent reacts rapidly (2 min) under mild conditions (pH 7.5, ambient) with thiol drugs and the resulting adducts can be chromatographed under reversed-phase conditions (C-18 column). Excess reagent can be removed from the reaction mixture by simple liquid-liquid extraction with chloroform. The HPLC method using UV detection was successfully applied to the analysis of reduced glutathione (GSH), N-acetylcysteine, N-2-mercaptopropionylglycine and captopril in their commercial formulations.

A novel automated hydrophilic interaction liquid chromatography method using diode-array detector/electrospray ionization tandem mass spectrometry for analysis of sodium risedronate and related degradation products in pharmaceuticals.

A simple, sensitive and fast hydrophilic interaction liquid chromatography (HILIC) method using ultraviolet diode-array detector (UV-DAD)/electrospray ionization tandem mass spectrometry was developed for the automated high performance liquid chromatography (HPLC) determination of sodium risedronate (SR) and its degradation products in new pharmaceuticals. The chromatographic separations were performed on Ascentis Express HILIC 2.7μm (150mm×2.1mm, i.d.) stainless steel column (fused core). The mobile phase consisted of formate buffer solution (pH 3.4; 0.03M)/acetonitrile 42:58 and 45:55 (v/v) for granules for oral solution and effervescent tablet analysis, respectively, at a flow-rate of 0.2mL/min, setting the wavelength at 262nm. Stability characteristics of SR were evaluated by performing stress test studies. The main degradation product formed under oxidation conditions corresponding to sodium hydrogen (1-hydroxy-2-(1-oxidopyridin-3-yl)-1-phosphonoethyl)phosphonate was characterized by high performance liquid chromatography-electrospray ionization-mass tandem mass spectrometry (HPLC-ESI-MS/MS). The validation parameters such as linearity, sensitivity, accuracy, precision and selectivity were found to be highly satisfactory. Linear responses were observed in standard and in fortified placebo solutions. Intra-day precision (relative standard deviation, RSD) was ≤1.1% for peak area and ≤0.2% for retention times (tR) without significant differences between intra- and inter-day data. Recovery studies showed good results for all the examined compounds (from 98.7 to 101.0%) with RSD ranging from 0.6 to 0.7%. The limits of detection (LOD) and quantitation (LOQ) were 1 and 3ng/mL, respectively. The high stability of standard and sample solutions at room temperature means an undoubted advantage of the method allowing the simultaneous preparation of many samples and consecutive chromatographic analyses by using an autosampler. The developed stability indicating method is suitable for the quality control of SR in new and commercial pharmaceutical formulations.

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).

A novel liquid chromatography method using diode-array detector for the determination of oleuropein in dietary supplements

A simple and fast chromatographic method using ultraviolet diode-array detector (UV-DAD) was developed for the automatic high performance liquid chromatography (HPLC) determination of the title of oleuropein in a new dietary supplements in form of effervescent granules. The chromatographic separations were performed on a C18 core-shell column with detection at λ=232nm. The mobile phase consisted of deionized water with 0.1% TFA and acetonitrile under gradient conditions at a flow-rate of 0.8mL/min. Oleuropein and oleuroside present in the raw material were characterized by high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). The validation of the analytical procedure has been performed determining the following parameters: specificity, linearity, repeatability, reproducibility, accuracy, limit of quantification (LOQ), stability of the standard and sample solutions. Linear response was observed in fortified placebo solutions (determination coefficient: 0.9998). Intra-day precision (relative standard deviation, RSD) was ≤5.0% for peak area and for retention times (tR) without significant differences between intra- and inter-day data. The limits of quantitation (LOQ) was about 5μg/mL and 9pmol/inject. Oleuropein recovery studies gave good results (99.9%) with a R.S.D. of 0.5%. The speed of analysis and the stability of the solutions with a fluctuation Δ (%) ≤2.0 at room temperature means an undoubted advantage of the method allowing the simultaneous preparation of many samples and consecutive chromatographic analyses by using an autosampler. The developed method is suitable for the quality control of oleuropein in raw material and industrial products. The method can be applied in any analytical laboratory not requiring a sophisticated instrumentation.

A novel hydrophilic interaction liquid chromatography method for the determination of underivatized amino acids in alimentary supplements

Graphical abstract Figure. No Caption available. HighlightsHILIC‐UV was applied to underivatized amino acids and amino acids‐like molecules.The method was validated and applied to dietary supplements.Oxidized glutathione was found as an impurity in some of the analysed real samples.RP‐HPLC based on derivatization of glutathione confirmed the results of HILIC. Abstract Amino acids playing important roles in metabolic processes are often included in dietary supplements whose use has largely expanded over the last 20 years not only in patients with particular deficiencies, but also in athletes and even common people that want to enrich their regular daily diet. In the present study, a bare silica Kinetex core‐shell 2.6 &mgr;m HILIC column was used for separation of some important hydrophilic amino acids and amino acids‐like molecules i.e., aspartic acid, creatine, carnitine, arginine and the tripeptide glutathione (GSH), by optimizing the chromatographic conditions for their determination in complex alimentary supplements. The contribution of partition, adsorption and ion exchange on the retention mechanism was studied by varying parameters such as water content and the counter‐ion concentration in the mobile phase. Optimum conditions employed a Phenomenex Kinetex core‐shell 2.6 &mgr;m HILIC (100 × 4.6 mm i.d.) column and a mobile phase of acetonitrile/potassium phosphate buffer (12.5 mM; pH = 2.8) 85:15, v/v, at the flow rate of 1.4 mL/min, using UV detection at 200 nm. A reference HPLC method for the selective determination of GSH by using 1,4‐naphthoquinone as derivatization reagent was also introduced for comparative purposes. The developed HILIC method was validated and applied to the analysis of the considered compounds in dietary supplements. Interestingly, in some of the real samples, oxidized glutathione which is an inactive impurity of GSH, was found at the level of about 20%. The proposed study confirms the importance of simple analytical methods for a rigorous quality control of dietary supplements containing unstable active ingredients.

1,4-Anthraquinone: A new useful pre-column reagent for the determination of N-acetylcysteine and captopril in pharmaceuticals by high performance liquid chromatography

&NA; 1,4‐Anthraquinone (ANQ) is proposed as a novel pre‐column reagent for high performance liquid chromatography (HPLC) determination of N‐acetylcysteine (NAC) and captopril (CAP) in pharmaceutical formulations. The derivatization reactions were carried out at room temperature: NAC at pH 8 for 1 min, while CAP at pH 7.5 for 20 min. Both reactions reached completeness at a reagent to thiol molar ratio of about 2.5. The synthesised derivatives were characterized by 1H NMR and IR. The chromatographic separations were performed on a C18 Phenomenex Synergi Fusion 4 &mgr;m (250 mm × 4.6 mm I.D.) stainless steel column with detection at &lgr; = 300 nm. The mobile phase consisted of methanol/triethylammonium (TEA) phosphate buffer (pH 3; 0.05 mol/L) 75:25 (v/v) at a flow‐rate of 0.4 mL/min for NAC and 88:12 (v/v), at a flow‐rate of 0.6 mL/min for CAP. The validation parameters (linearity, sensitivity, accuracy, precision, specificity and stability) were highly satisfactory. Linear response was observed (determination coefficient ≥0.9996). Detection limits were about 8 and 18 ng/mL for NAC and CAP, respectively. Intra‐day precision (relative standard deviation, R.S.D.) was ≤1.58%, for thiol to internal standard (IS) peak area ratio and ≤0.33%, for thiol and IS retention times (tR), without significant differences between intra‐ and inter‐day data. Thiol recovery studies were satisfactory (99.50%) with R.S.D. ≤0.56%. The results highlight the high sensitivity of the method and the remarkable reactivity and selectivity of the reagent towards the thiol function. The developed method is suitable for the quality control of both thiols in commercial products. The method can be applied in any analytical laboratory not requiring a sophisticated instrumentation. Graphical abstract Figure. No caption available. HighlightsN‐Acetylcysteine (NAC) and captopril (CAP) were derivatized with antraquinone (ANQ).ANQ is a novel sensitive reagent suitable for thiol HPLC analysis.Derivatization reactions were carried out in short time at room temperature.NAC and CAP derivatives were synthesized and characterized by 1H NMR and IR.The method is easy to apply not requiring a sophisticated instrumentation.

Simple Determination of 1-Deoxynojirimycin in a New Dietary Supplement by Liquid Chromatography

A simple and fast chromatographic method using ultraviolet diode-array detector (UV-DAD) was developed for the high performance liquid chromatography determination of the content of 1-deoxynojirimycin (DNJ) in a new dietary supplement in thexa0form of granules for oral solutionxa0preparation. The derivatization reaction was carried out at room temperature for 15xa0min at pH 7. The reaction reached completeness at a reagent to analyte molar ratio of about 60. The chromatographic separations were performed on a C18 Phenomenex Synergi Fusion stainless steel column (250xa0mmu2009×u20094.6xa0mm; 4xa0µm) with detection at λu2009=u2009254xa0nm. The mobile phase consisted of triethylammonium (TEA) phosphate buffer (0.05xa0M; pH 3) and acetonitrile under gradient conditions at a flow-rate ramping from 1 to 1.2xa0mL/min. The validation parameters (linearity, sensitivity, accuracy, precision, specificity and stability) were satisfactory. Intra-day precision (relative standard deviation, RSD) was ≤u20092.23% for peak area and retention time without significant differences between intra- and inter-day data. Recovery studies gave good results (93.59%; nu2009=u200915) with a RSD of 2.64%. The developed method is suitable for the quality control of DNJ in raw material and industrial products. The method can be applied in any analytical laboratory and does not require sophisticated instrumentation.