Synthetic antisense phosphorothioate oligonucleotides (PS) have undergone rapid development as novel therapeutic agents. The increasing significance of this class of drugs requires significant ...investment in the development of quality control methods. The determination of the many degradation pathways of such complex molecules presents a significant challenge. However, an understanding of the potential impurities that may arise is necessary to continue to advance these powerful new therapeutics. In this study, four different antisense oligonucleotides representing several generations of oligonucleotide therapeutic agents were evaluated under various stress conditions (pH, thermal, and oxidative stress) using ion-pairing reversed-phase liquid chromatography tandem mass spectrometry (IP-RPLC-MS/MS) to provide in-depth characterization and identification of the degradation products. The oligonucleotide samples were stressed under different pH values at 45 and 90 °C. The main degradation products were observed to be losses of nucleotide moieties from the 3′- and 5′-terminus, depurination, formation of terminal phosphorothioates, and production of ribose, ribophosphorothioates (Rp), and phosphoribophosphorothioates (pRp). Moreover
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the effects of different concentrations of hydrogen peroxide were studied resulting in primarily extensive desulfurization and subsequent oxidation of the phosphorothioate linkage to produce the corresponding phosphodiester. The reaction kinetics for the degradation of the oligonucleotides under the different stress conditions were studied and were found to follow pseudo-first-order kinetics. Differences in rates exist even for oligonucleotides of similar length but consisting of different sequences.
Graphical abstract
Identification of degradation products across several generations of oligonucleotide therapeutics using LC-MS
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.
Two accurate, reliable, and highly sensitive spectrofluorometric methods were developed for simultaneous determination of the binary mixture of Atorvastatin and Ezetimibe without prior separation ...steps. The first method is based on double scan synchronous fluorescence spectrometry. Each of Atorvastatin and Ezetimibe can be determined independent of the other when scanned at Δλ=100 nm and 40 nm, respectively. The relative fluorescence intensity–concentration plots at two wavelengths, 272 (Δλ=100 nm) and 266 nm (Δλ=40 nm) were rectilinear over the range of 0.4–8 µg/mL (for Atorvastatin) and 0.6–8 µg/mL (for Ezetimibe), respectively. The second method is based on the technique of simultaneous equations (Vierodt’s method), in which two equations are solved simultaneously after using a single excitation wavelength of 273 nm and λEm1=380 nm of Atorvastatin and λEm2=301 nm of Ezetimibe. Under the optimum conditions, linear relationships were found between the relative fluorescence intensity and the concentrations of the investigated drugs in the range of 0.4–8 µg/mL (for Atorvastatin) 0.6–8 µg/mL (for Ezetimibe). The different experimental parameters affecting the fluorescence intensities of the two drugs were carefully studied and optimized. The proposed methods were successfully applied for the determination of the investigated drugs in pure form, dosage form and in synthetic mixtures with good recovery and the results obtained were favorably compared to those obtained with a reference method.
Recently functional polymers have attracted significant attention in the area of pharmaceuticals and biomedical applications, so it is important to develop simple techniques to analyze functional ...polymers in their pharmaceutical dosage forms.
: Three simple, accurate and sensitive UV spectrophotometric methods have been developed and validated for determination of Polyvinyl pyrrolidone (PVP) in the presence of benzalkonium chloride (BZ) and sodium lactate in ternary mixture.
Method A is derivative ratio spectra zero-crossing (DRSZ) method measures PVP peak amplitude at 303.1 nm. Method B is a double divisor ratio derivative (DDRD), used for determination of both PVP and BZ in the presence of sodium lactate at 272.6 and 271.5 nm, respectively. Method C is double divisor ratio derivative-Ratio difference spectrophotometric method (DDRD-RDSM), a new and hybrid method of double divisor and ratio difference that hasn't been applied before, it measures peak amplitude difference of the ratio spectra at (ΔP278- 252.4) and (ΔP260.9-213) for PVP and BZ; respectively.
Linear range for PVP (5.00-35.00 µg/mL),(10.00-40.00 µg/mL) and (10.00-40.00 µg/mL) was obtained by using (DRSZ), (DDRD) and (DDRD-RDSM); respectively. While linear range for BZ (5.00-60 µg/mL) was obtained by using both (DDRD) and (DDRD-RDSM).
All results were statistically compared with reported methods, no significant difference was observed. The developed methods were applied to the analysis of the investigated drugs in pure and pharmaceutical dosage forms.
The proposed methods are of great value to be used efficiently for routine analysis of PVP and BZ in their pharmaceutical dosage forms.
Abstract
Egypt has the highest prevalence of hepatitis C virus (HCV) in the world thus it launched a national program for eliminating HCV aiming to treat 300,000 HCV patients per year. Three anti-HCV ...co-administered drugs; ribavirin (RBV), sofosbuvir (SF) daclatasvir (DAC) were simultaneously determined in human plasma by a validated, simple and sensitive RP-HPLC method using propyl paraben as an internal standard. Liquid–liquid extraction using ethyl acetate was used for samples extraction. Chromatographic separation was achieved on Scharlau® C18 column (250 × 4.6 mm2, 5 μm). Gradient elution was employed with a mobile phase mixture of water and acetonitrile at a flow rate 1 mL/min. UV detection using photodiode array detector was carried out at 207, 260 and 312 nm for RBV, SF and DAC, respectively. Method validation was performed according to the FDA guidelines for bioanalytical method validation. The calibration curves were linear over the ranges (0.5–80, 0.1–40 and 0.5–80 μg/mL) with average recoveries (100.64–108.28%, 98.48–105.91% and 97.68–101.38%) for RBV, SF and DAC, respectively. The intra-day and inter-day precision and accuracy results were within the acceptable limits. Stability assays revealed that the three studied analytes were stable during sample storage, preparation and injection. The method can be successfully applied in routine analysis of plasma of HCV patients treated with this combination therapy which aids in therapeutic drug monitoring and patients’ follow-up especially in Egypt and other developing countries fighting HCV.
Chitosan (CH) is one of the most abundant biopolymers with multiple applications. Polyvinyl pyrrolidone (PVP) has specific binding and detoxification properties that are of great interest in health ...care. Hence, it arises a crucial urge to develop economic sensors to analyze CH and PVP in pharmaceutical formulations and biological samples. Two portable sensors were fabricated using precipitation‐based technique, and nanoparticles‐based technique, for determination of CH and PVP in sensor 1 and 2; respectively. Linear responses of 10−5 to10−7 M and 10−2 to10−7 M at pH 3.6–4.8 and 7.2–8.4, with ideal Nernstian slopes of 60.00 and 59.83 mV /decade, and nanomolar LODs of 94.90 and 81.20 nM were observed for CH and PVP; respectively. The percentage recoveries were 100.40±1.03 and 100.19±0.64 for sensors 1 and 2; respectively. Both sensors were successfully applied in biological fluids without pre‐treatment. Accurate results were obtained using sensor 1, in pure form, pharmaceutical formulations, human plasma, rat liver and rat brain, as well as sensor 2, in pure form, pharmaceutical formulations and urine samples. The results were statistically compared with the reported methods and no significant difference was observed.
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•These methods are sensitive and resolve binary mixture with overlapping spectra.•The good recovery and accuracy make them applicable in QC laboratories.•The proposed methods can be ...used in QC without the difficulties of HPLC.•The proposed methods were validated according to ICH guidelines.•The proposed methods have been applied successfully on veterinary formulation.
Five simple, specific, accurate and precise UV-spectrophotometric methods are adopted for the simultaneous determination of Amprolium hydrochloride (AMP) and Ethopabate (ETH), a binary mixture with overlapping spectra, without preliminary separation. The first method is first derivative of the ratio spectra (1DD) for determination of AMP and ETH at 234.7nm and 306.8nm respectively with mean percentage recoveries 99.76±0.907 and 100.29±0.842 respectively. The second method is the mean centering of the ratio spectra for determination of AMP and ETH at 238.8nm and 313nm respectively with mean percentage recoveries 100.26±1.018 and 99.94±1.286 respectively. The third method is based on dual wavelength selection for determination of AMP and ETH at 235.3nm & 308nm and 244nm & 268.4nm respectively with mean percentage recoveries 99.30±1.097 and 100.03±1.065 respectively. The fourth method is ratio difference method for determination of AMP and ETH at 239nm & 310nm and 239nm & 313nm respectively with mean percentage recoveries 99.27±0.892 and 100.40±1.814 respectively. The fifth one is area under the curve (AUC) method where the areas between 235.6–243nm and 268.3–275nm are selected for determination of AMP and ETH with mean percentage recoveries 100.35±1.031 and 100.39±0.956 respectively. These methods are tested by analyzing synthetic mixtures of the two drugs and they are applied to their pharmaceutical veterinary preparation. Methods are validated according to the ICH guidelines and accuracy, precision and repeatability are found to be within the acceptable limit.
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 293.0nm, 284.0nm, 276.0nm, 257.0nm and 221.0nm within linear concentration ranges 3.00–45.00, 1.00–15.00, 4.00–40.00, 1.50–25.00 and 4.00–50.00μgmL−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 4.00–12.00μgmL−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.
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•Spectrophotometric and chemometric methods were developed and validated.•Principal component regression and partial least-squares done for this mixture•Extended derivative ratio developed for this combination•These methods are sensitive and resolve mixtures with severely overlapping spectra.•The good recovery and accuracy make them applicable in QC laboratories.
UV spectroscopy of tramadol (TRA) and paracetamol (PAR) shows substantial spectral overlap, which is a challenge for their simultaneous determination without preliminary separation. Three smart ...spectrophotometric methods based on the ratio spectra developed from the overlapping UV spectra of their binary mixture can be applied for a quantitative estimation of both drugs. The first derivative (DR
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) of the ratio spectra was computed, and then the amplitudes were measured at 268.7 and 237.4 nm for TRA and PAR, respectively. The mean centered ratio (MCR) of the spectra was estimated by measuring the MCR spectra at 279 and 241.5 nm for TRA and PAR, respectively. Finally, the dual wavelength (DW) method was applied by measuring the difference in absorbance at 224.1 and 268.5 nm for TRA determination and at 248 and 285.4 nm for PAR determination without any interference. All the above-mentioned spectrophotometric methods can be used to estimate TRA in the linear range of 10–110 μg/mL. Furthermore, PAR can be estimated in the linear range of 1–25 μg/mL. These methods were successfully applied to the analysis of the combined dosage form and bulk powder of TRA and PAR. The methods were validated according to the International Conference on Harmonization (ICH) guidelines, and the obtained results were statistically compared with those obtained by previously reported methods. No significant difference with respect to accuracy and precision was observed.
Microtubules (MTs) play important and diverse roles in eukaryotic cells. Their function and biophysical properties have made alpha-and beta-tubulin, the main components of MTs, the subject of intense ...study. Interfering with normal MT dynamics, for example, by the addition of tubulin ligands, can cause the cell great distress and affect MT stability and functions, including mitosis, cell motion and intracellular organelle transport. It has been shown in the literature that tubulin is an important target molecule for developing anticancer drugs. Tubulin binding molecules have generated considerable interest after the successful introduction of the taxanes into clinical oncology and the widespread use of the vinca alkaloids vincristine and vinblastine. These compounds inhibit cell mitosis by binding to the protein tubulin in the mitotic spindle and preventing polymerization into the MTs. This mode of action is also shared with other natural agents eg colchicine and podophyllotoxin. However various tubulin isotypes have shown resistance to taxanes and other MT agents. Therefore, there is a strong need to design and develop new natural analogs as antimitotic agents to interact with tubulin at sites different from those of vinca alkaloids and taxanes. This minireview provides SAR on several classes of antimitotic agents reported in the literature. The structures and data given are essential to the scientists who are involved in drug design and development in the field of anticancer drugs.
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