HPLC method development for fampridine using Analytical Quality by Design approach.

Offering a systematic and multivariate analysis of the analytical procedure, development and validation of HPLC methods using Quality by Design approach are in the limelight of current research trends. A new, experimental design-aided HPLC method for fampridine was developed and preliminarily validated according to current in-force international guidelines for linearity, accuracy, robustness and precision. The method offers a high throughput sample analysis, with an elution time of 2.9 minutes, and signal detection without excipient interference performed at 262 nm. The method proved to be linear between 1-15 µg mL-1 (R2= 0.9996). The mean recovery was found to be 98.7 ± 1.9 % in the tested range of 2.5-7.5 µg mL-1. Low RSD values (< 1 %) were obtained for both model, intra- and inter-day precision. The limit of detection and limit of quantification were 0.24 and 0.78 µg mL-1, resp. The method proved to be applicable for active substance assay in a pharmaceutical dosage form.

Graphene nanoribbon/FePt bimetallic nanoparticles/uric acid as a novel magnetic sensing layer of screen printed electrode for sensitive determination of ampyra.

A novel electrochemical sensor for sensitive determination of ampyra (Am) based on graphene nanoribbons modified by iron-platinum bimetallic nanoparticles and uric acid (SPCE/FePtGNR/UA) dropped on the screen-printed carbon electrode (SPCE) surface and magnetically captured onto an SPCE working electrode surface is reported in the present work. The modified nanocomposite and sensing layer was characterized by different techniques, including cyclic voltammetry (CV), linear sweep voltammetry (LSV), electrochemical impedance spectroscopy (EIS), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR) and X-ray powdered diffraction (XRD). Am determination by conventional electrochemical methods is not possible, because of its high redox overpotential. Therefore, the differential pulse voltammetry (DPV) signals of UA were used as a redox probe for indirect electrochemical determination of Am. The limit of detection (LOD) and linear concentration range were obtained as 0.028 and 0.08-9.0µmolL-1 (3Sb/m = 3), respectively. The feasibility of the proposed method was examined by the detection of Am in biological and pharmaceutical samples with satisfactory results. The constructed electrochemical sensor was applied for fast, simple and sensitive detection of Am in real environments.

Fabrication of a novel impedimetric sensor based on l-Cysteine/Cu(II) modified gold electrode for sensitive determination of ampyra.

This paper presents a novel impedimetric sensor for the sensitive determination of ampyra (Am) based on the l-Cysteine/Cu (II) modified gold electrode (Cu (II)/L-Cys/AuE). This novel sensing layer was characterized by different techniques, including atomic force microscopy (AFM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Am determination by conventional electrochemical methods is not possible, because of its high redox overpotential. Therefore, [Fe(CN)6]3-/4- was used as a redox probe for the impedimetric determination of Am. Under the optimized conditions, the calibration curve for Am concentration was linear in the range 0.010-40.0 nM with a detection limit of 0.0028 nM. The practical applicability of the proposed sensor was examined by evaluating the detection of Am in biological fluids and pharmaceutical samples with satisfied recoveries. Therefore, the prepared sensor can hold great promise for fast, simple and sensitive detection of Am in various real samples.

A new derivatization reagent for LC-MS/MS screening of potential genotoxic alkylation compounds.

A screening method for trace analysis of potentially genotoxic alkylating compounds has been developed using butyl 1-(pyridin-4-yl) piperidine 4-carboxylate (BPPC) as a new, selective pre-column derivatization reagent for their subsequent analysis by hydrophilic interaction liquid chromatography (HILIC) hyphenated with tandem mass spectrometry (LC-MS/MS). The new derivatization reagent is a modification of 4-dimethylaminopyridine (4-DMAP) previously used for the determination of potentially genotoxic compounds. By using the new reagent the screening potential was enhanced without compromising reactivity. Derivatization at a high pH value was carried out and the reaction time at 60°C was 24h to anticipate for alkyl chlorides showing to be less reactive. The new reagent was designed to obtain reagent related fragmentation of the whole reagent as well as a side group of the reagent. Collision energies for detection of alkylating components derivatized using the new reagent are shown to be significantly more universal than with 4-DMAP. Neutral loss scanning on the fragmentation related to the build in side group remedies shortcomings in the screening for alkyl halides observed when using 4-DMAP. The new approach allows for screening of alkyl halides and alkyl sulfonates at trace levels down to 1 mg kg(-1) and target analysis at about a factor of 10 lower without a significant effect of the active pharmaceutical ingredient (API) matrix. The synthesis of the reagent, investigation of reactivity, the specificity of the fragmentation of derivatives and screening conditions in MS/MS analysis are described.

Design of experiments as a tool for LC-MS/MS method development for the trace analysis of the potentially genotoxic 4-dimethylaminopyridine impurity in glucocorticoids.

The present study reports on a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method development strategy supported by design of experiments (DoE) for the trace analysis of 4-dimethylaminopyridine (DMAP). The conventional approaches for development of LC-MS/MS methods are usually via trial and error, varying intentionally the experimental factors which is time consuming and interactions between experimental factors are not considered. The LC factors chosen for the DoE study include flow (F), gradient (G) and injection volume (V(inj)) while cone voltage (E(con)) and collision energy (E(col)) were chosen as MS parameters. All of the five factors were studied simultaneously. The method was optimized with respect to four responses: separation of peaks (Sep), peak area (A(peak)), length of the analysis (T) and the signal to noise ratio (S/N). A quadratic model, namely central composite face (CCF) featuring 29 runs was used instead of a less powerful linear model since the increase in the number of injections was insignificant. In order to determine the robustness of the method a new set of DoE experiments was carried out applying robustness around the optimal conditions was evaluated applying a fractional factorial of resolution III with 11 runs, wherein additional factors - such as column temperature and quadrupole resolution - were considered. The method utilizes a Phenomenex Gemini NX C-18 HPLC analytical column with electrospray ionization and a triple quadrupole mass detector in multiple reaction monitoring (MRM) mode, resulting in short analyses with a 10min runtime. Drawbacks of derivatization, namely incomplete reaction and time consuming sample preparation, have been avoided and the change from SIM to MRM mode resulted in increased sensitivity and lower LOQ. The DoE method development strategy led to a method allowing the trace analysis of DMAP at 0.5 ng/ml absolute concentration which corresponds to a 0.1 ppm limit of quantification in 5mg/ml mometasone furoate glucocorticoid. The obtained method was validated in a linear range of 0.1-10 ppm and presented a %RSD of 0.02% for system precision. Regarding DMAP recovery in mometasone furoate, spiked samples produced %recoveries between 83 and 113% in the range of 0.1-2 ppm.

Development of more potent 4-dimethylaminopyridine analogues.

[reaction: see text] The syntheses of bicyclic diaminopyridines 3 and 4 and tricyclic triaminopyridines 5 and 6, two novel series of nucleophilic catalysts, are described. Arguments are made for predicting the superiority of these catalysts over DMAP and even 2, the best esterification catalyst reported to date. The efficiencies of DMAP, PPY, and 2-6 in catalyzing the esterification of tertiary alcohols were compared. As predicted, 5 and 6 were about 6-fold more effective than DMAP and slightly better than 2.

Stability studies of ionised and non-ionised 3,4-diaminopyridine: hypothesis of degradation pathways and chemical structure of degradation products.

3,4-Diaminopyridine is used to treat some symptoms met in Lambert-Eaton myasthenia syndrome. It was shown efficient to reduce a form of variable muscle weakness and fatigability typical of the disease and correlated to a block of acetylcholine release. In France, 3,4-diaminopyridine is nowadays given to patients under capsules form and the status of hospital preparation. Whatever the diluant used in the formulation, the stability period could not exceed 12 months. Preliminary studies were made on a salt form in order to test the influence of various stress factors and determine if there is interaction between them. From this study, the most influent stress condition, presence of hydrogen peroxide, was selected and a comparative study was performed to compare the stability of molecular and salt species. Solutions of each species were exposed to 5 or 15% of hydrogen peroxide and analyzed at 8, 24, 72 and 216 h of degradation by HPLC-UV. Fractions of detected impurities were purified and collected by semi-preparative HPLC-UV and analyzed by HPLC-UV-ESI-MS and IR spectroscopy in order to determine their structure hypotheses. Theses experiments demonstrate that the salt species were more stable under oxidative stress condition than molecular species. The two main degradation products were collected and identified as 4-amino, 3-nitropyridine and 3,4-diaminopyridine-N-oxide when the molecular form was degraded whereas only 4-amino, 3-nitropyridine was found in less quantity in the salt solutions. Nitrogen pyridine and pyridine amine could not easily be oxidized by hydrogen peroxide in salt comparatively to molecular species due to the lone pair of electron engaged in a bound with hydrogen in the first case and by resonance change of the pyridine in the second case. This modification of structure promoted different pathways of degradation for the salt form which are more dependent of energy. Owing to the better stability of the salt species, a new pharmaceutical form containing it was developed to assess its stability under ICH standard conditions allowing an industrial manufacture of this drug.

Validation of a CE assay for the analysis of isomeric aminopyridines and diaminopyridines.

A capillary electrophoresis assay for the analysis of aminopyridines and diaminopyridines has been developed and validated. The compounds were separated using a 100 mM sodium acetate buffer at pH 5.15, an applied voltage of 20 kV and a capillary of 60 cm effective length, N-(1-naphthyl)ethylenediamine was used as internal standard to compensate for injection errors and minor fluctuations of the migration times. The detection wavelength was set at 240 nm and not optimized for a specific derivative. The assay was validated with respect to specificity, linearity, range, limit of quantitation and detection, precision, and robustness. Within certain limits, the assay also allowed the detection and determination of the other aminopyridine derivatives at the 0.1% level as demonstrated for 3,4-diaminopyridine.

Development and validation of a capillary electrophoresis assay for the determination of 3,4-diaminopyridine and 4-aminopyridine including related substances.

A capillary electrophoresis (CE) assay has been developed for the quantitation and determination of the impurity profile of the potassium channel blockers 3,4-diaminopyridine and 4-aminopyridine. The compounds were separated from related substances using a capillary of 30 cm effective length, a 50 mM phosphate buffer, pH 2.5 and an applied voltage of 25 kV. The assay was validated with respect to specificity, linearity, range, limits of quantitation and detection, precision and robustness. The method allows the detection and quantitation of impurities at the 0.05% level. The feasibility of the assay was demonstrated by analyzing a commercial sample of 3,4-diaminopyridine. All known related substances could be detected in this sample with the present CE method.

Optimization of the determination of 4-aminopyridine in human serum and urine by column liquid chromatography.

Two convenient reversed-phase column liquid chromatographic procedures are described for the determination of 4-aminopyridine in human serum and urine. A 0.5-ml aliquot of serum after the addition of a 0.5-ml solution of 4-(aminomethyl)pyridine in 0.1 M Na2HPO4 as the internal standard is passed through a 1-ml BondElut C18 silica extraction column. The column is selectively washed to remove acidic, neutral and weakly basic compounds. The desired compounds are eluted with a 0.3-ml aliquot of 35% perchloric acid-methanol (1:100, v/v). A 10-microliters aliquot of the eluate is injected onto a 150 x 4.6 mm I.D. column packed with 5-microns C18 silica particles that is eluted at ambient temperature with a mobile phase containing octanesulfonic acid as the ion-pairing agent. The peaks are monitored at 263 nm. A. 0.25-ml aliquot of urine or 0.5 ml of serum is mixed with N-propionylprocainamide as the internal standard and subjected to benzoylation by Schotten Baumann reaction. The reaction mixture is adjusted to pH 5.5-6 and extracted with a BondElut C18 extraction column. An aliquot of the eluate is chromatographed at ambient temperature with a mobile phase containing tetramethylammonium perchlorate. The peaks are monitored at 278 nm.

Polymeric dimethylaminopyridinium reagents for derivatization of weak nucleophiles in high-performance liquid chromatography-ultraviolet/fluorescence detection.

This paper introduces a novel polymeric dimethylaminopyridinium 9-fluorenyl-methoxycarbonyl reagent for off-line derivatizations of weak nucleophiles in high-performance liquid chromatography. The method of synthesis and characterization of the polymeric reagent via loading determinations is presented and discussed. Derivatization conditions (solvent, time, and temperature) for primary and secondary alcohols were optimized. As one application, off-line derivatizations of 2-chloro-1-propanol, a potential carcinogen in foodstuffs, were carried out with this polymeric reagent with single-blind and standard addition techniques. A specific sample treatment procedure was also developed. The accuracy and precision of the method were determined and data were statistically evaluated.

Sensitive trace-first liquid chromatographic determination of 4-aminopyridine in 3,4-diaminopyridine.

For the treatment of human neuromuscular diseases, 3,4-diaminopyridine (DAP) is six to ten times more effective than 4-aminopyridine (AP), but only half as convulsant and toxic. Therefore, there is a need for the determination of AP in DAP. With only conventional equipment, high-pressure liquid chromatography can be used for the extremely sensitive detection of a trace contaminant under one condition: that is, the trace must be eluted before the major component it contaminates. Prior elution presents a trace peak in a fully exploitable form that is maximally efficient and maximally observable. This has already been demonstrated with a Pirkle-concept chiral stationary phase for determination of a chiral trace. However, its application to determination of a nonchiral trace with a reversed phase has not previously been reported. Such an application is reported here. In this demonstrative study, selectivity and loading capability were iteratively improved. Ion pairing with dodecanesulfonate maximized selectivity. It was again shown that using a less concentrated sample in greater volume maximizes loading capability without obscuring the peak of the trace. Eventually, the ability to detect 0.005% AP in DAP was demonstrated. Whether that sensitivity might be improved still more, perhaps with a larger column, was not established.

A high-performance liquid chromatography method for determination of 4-aminopyridine in tissues and urine.

An analytical method was developed to measure 4-aminopyridine in tissues and urine to determine appropriate diagnostic samples in acute poisoning cases. Tissues from rats dosed with 4-aminopyridine were extracted with methylene chloride. Extracts were analyzed by high-performance liquid chromatography using an isocratic solvent system of acetonitrile and aqueous solution (15/85 v/v) consisting of 0.015 M sodium salt of l-heptane-sulfonic acid, 0.002 M tetramethylammonium bromide, and 0.01 M sodium dihydrogen phosphate adjusted to pH 3.0 with phosphoric acid. We concluded that suitable diagnostic samples for acute poisoning cases include stomach contents, kidney, liver, and urine.