Alkylamine ion-pairing reagents and the chromatographic separation of oligonucleotides.

Alkylamines are commonly used to improve both chromatographic and mass spectral performance of electrospray ionization liquid chromatography mass spectrometry based methods for the analysis of oligonucleotides. Recently several new alkylamines have been introduced to enhance the electrospray mass spectral response for oligonucleotides; however, the chromatographic properties of these new alkylamines have not been rigorously assessed. We have investigated the retention, peak width, resolution and general chromatographic performance of fifteen different alkylamines for the separation of a model DNA, RNA and an antisense therapeutic oligonucleotide. Eleven of the fifteen alkylamines were shown to provide similar chromatographic performance across all three classes of oligonucleotides. Based on these findings, a model for the mechanism of retention of oligonucleotides using alkylamines and hexafluoroisopropanol mobile phases is proposed. Depending on the concentrations of alkylamines and pH adjustment, oligonucleotides can be retained by micellar chromatography and not the generally held ion-pairing mechanism. This conclusion is supported by light scattering, transmission electron microscopy and ion mobility experiments detecting three micron aggregates in the mobile phase at concentrations that are routinely used for LC-MS analysis of oligonucleotides. These aggregates are not detected at lower alkylamine concentrations where the retention mechanism follows an ion-pairing mechanism. The formation of these aggregates appears to be dependent on the pH of the mobile phase.

Chromatographic approaches for the characterization and quality control of therapeutic oligonucleotide impurities.

Phosphorothioate (PS) oligonucleotides are a rapidly rising class of drugs with significant therapeutic applications. However, owing to their complex structure and multistep synthesis and purification processes, generation of low-level impurities and degradation products are common. Therefore, they require significant investment in quality control and impurity identification. This requires the development of advanced methods for analysis, characterization and quantitation. In addition, the presence of the PS linkage leads to the formation of chiral centers which can affect their biological properties and therapeutic efficiency. In this review, the different types of oligonucleotide impurities and degradation products, with an emphasis on their origin, mechanism of formation and methods to reduce, prevent or even eliminate their production, will be extensively discussed. This review will focus mainly on the application of chromatographic techniques to determine these impurities but will also discuss other approaches such as mass spectrometry, capillary electrophoresis and nuclear magnetic resonance spectroscopy. Finally, the chirality and formation of diastereomer mixtures of PS oligonucleotides will be covered as well as approaches used for their characterization and the application for the development of stereochemically-controlled PS oligonucleotides.

Spectrophotometric and chemometric methods for determination of imipenem, ciprofloxacin hydrochloride, dexamethasone sodium phosphate, paracetamol and cilastatin sodium in human urine.

New accurate, sensitive and selective spectrophotometric and chemometric methods were developed and subsequently validated for determination of Imipenem (IMP), ciprofloxacin hydrochloride (CIPRO), dexamethasone sodium phosphate (DEX), paracetamol (PAR) and cilastatin sodium (CIL) in human urine. These methods include a new derivative ratio method, namely extended derivative ratio (EDR), principal component regression (PCR) and partial least-squares (PLS) methods. A novel EDR method was developed for the determination of these drugs, where each component in the mixture was determined by using a mixture of the other four components as divisor. Peak amplitudes were recorded at 293.0 nm, 284.0 nm, 276.0 nm, 257.0 nm and 221.0 nm within linear concentration ranges 3.00-45.00, 1.00-15.00, 4.00-40.00, 1.50-25.00 and 4.00-50.00 µg mL(-1) for IMP, CIPRO, DEX, PAR and CIL, respectively. PCR and PLS-2 models were established for simultaneous determination of the studied drugs in the range of 3.00-15.00, 1.00-13.00, 4.00-12.00, 1.50-9.50, and 4.00-12.00 µg mL(-1) for IMP, CIPRO, DEX, PAR and CIL, respectively, by using eighteen mixtures as calibration set and seven mixtures as validation set. The suggested methods were validated according to the International Conference of Harmonization (ICH) guidelines and the results revealed that they were accurate, precise and reproducible. The obtained results were statistically compared with those of the published methods and there was no significant difference.

Screening and Optimization of the Reaction of Polymyxin B Sulphate with NBD-Cl for the Synchronous Spectrofluorimetric Determination of Polymyxin B Sulphate in Human Plasma.

An accurate and sensitive synchronous spectrofluorimetric method has been developed for the determination of Polymyxin B sulphate (Poly B) in human plasma. The method is based on the reaction of non-fluorescent Poly B with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) in borate buffer of pH 7 producing a yellow color with maximum relative fluorescence at 440 nm using a constant wavelength difference Δλ = 80 nm. Reaction conditions and other analytical parameters were studied and optimized using factorial design. Three level factorial designs have been employed for the screening, optimization of all experimental variables and determination of their interactions on the final product formation. The variables under investigation were: pH of borate buffer, volume of buffer, volume of NBD-Cl, temperature, time of heating and volume of sulfuric acid. A linear plot between relative fluorescence and concentration was obtained over the concentration range 100.00-1200.00 ng mL(-1). The limit of detection (LOD) and limit of quantification (LOQ) were found to be 10.31 and 31.24 ng mL(-1), respectively. The proposed method was validated according to ICH guidelines and successfully applied for the determination of Poly B in human plasma, where satisfactory results were obtained. The results obtained were statistically compared with those of a published method, where no significant difference was observed.

Novel spectroscopic methods for determination of Cromolyn sodium and Oxymetazoline hydrochloride in binary mixture.

New accurate, sensitive and selective spectrophotometric and spectrofluorimetric methods were developed and subsequently validated for determination of Cromolyn sodium (CS) and Oxymetazoline HCl (OXY) in binary mixture. These methods include 'H-point standard addition method (HPSAM) and area under the curve (AUC)' spectrophotometric method and first derivative synchronous fluorescence spectroscopic (FDSFS) method. For spectrophotometric methods, absorbances were recorded at 241.5nm and 274.9nm for HPSAM and the wavelength was selected in ranges 232.0-254.0nm and 216.0-229.0nm for AUC method, where the concentration was obtained by applying Cramer's rule. For FDSFS method, the first-derivative synchronous fluorescence signal was measured at 290.0nm, using Δλ=145.0nm. The suggested methods were validated according to International Conference of Harmonization (ICH) guidelines and the results revealed that they were precise and reproducible. All the obtained results were statistically compared with those of the reported method and there was no significant difference.