Quantitation of memantine hydrochloride bulk drug and its tablet formulation using proton nuclear magnetic resonance spectrometry

The use of quantitative nuclear magnetic resonance spectrometry for the determination of non‐UV active memantine hydrochloride with relative simplicity and precision has been demonstrated in this study. The method was developed on a 500 MHz NMR instrument and was applied to determination of the drug in a tablet formulation. The analysis was performed by taking caffeine as an internal standard and D₂O as the NMR solvent. The signal of methyl protons of memantine hydrochloride appeared at 0.75 ppm (singlet) relative to the signal of caffeine (internal standard) at 3.13 ppm (singlet). The method was found to be linear (r² = 0.9989) in the drug concentration range of 0.025 to 0.80 mg/ml. The maximum relative standard deviation for accuracy and precision was <2. The limits of detection and quantification were 0.04 and 0.11 mg/ml, respectively. The robustness of the method was revealed by changing nine different parameters. The deviation for each parameter was also within the acceptable limits. The study highlighted possibility of direct determination of memantine hydrochloride in pure form and in its marketed tablet formulation by the use of quantitative NMR, without the need of derivatization, as is the requirement in HPLC studies. Copyright © 2016 John Wiley & Sons, Ltd.     

Purity determination of a new antifungal drug candidate using quantitative 1H NMR spectroscopy: Method validation and comparison of calibration approaches

Quantitative nuclear magnetic resonance (qNMR) is an analytical technique that offers numerous advantages in pharmaceutical applications including minimum sample preparation and rapid data collection times with no need for response factor corrections, being a powerful tool for assaying drug content in both drug discovery and early drug development. In the present work, we have applied qNMR, using both the internal standard and the electronic reference to access in vivo concentrations 2 calibration methods, to assess the purity of RI76, a novel antifungal drug candidate. NMR acquisition and processing parameters were optimized in order to obtain spectra with intense, well-resolved signals of completely relaxed nuclei. The analytical method was validated following current guidelines, demonstrating selectivity, linearity, accuracy, precision, and robustness. The calibration approaches were statistically compared, and no significant difference was observed when comparing the obtained results and their dispersion in terms of relative standard deviation. The proposed qNMR method may, therefore, be used for both qualitative and quantitative assessments of RI76 in early drug development and for characterization of this compound.     

Optimization and validation of stability indicating RP-HPLC method for the quantification of gefitinib in bulk drug and nanoformulations: An application towards in vitro and ex vivo performance evaluation

Gefitinib (GFB) is an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor used primarily to treat non-small cell lung cancer (NSCLC), but it also works by lowering AKT and MAPK phosphorylation, which makes it effective against the breast cancer cells. A high-performance liquid chromatography (HPLC) method was developed for detecting gefitinib by C18 analytical column with mobile phase containing acetonitrile and 1 % w/v ammonium acetate in water with ratio of 60:40. Box-Behnken design was used to optimize the chromatographic conditions, and method was validated for accuracy, precision, linearity, robustness, ruggedness, limit of detection, and limit of quantification. The developed method was found to be useful in estimating gefitinib in conventional marketed tablet dosage forms and nanoformulations such as SLNs and liposomes. With a calibration curve, linearity was attained in the drug concentration range of 8–56 µg/mL (r² = 0.9996), and sensitivity was found to be 1.3 and 3.9 µg/mLas LOD and LOQ, respectively. All the validation criteria for the method were within the acceptable limits. The drug content in the conventional tablet formulation was found to be 99.99 %. The HPLC analysis indicated that gefitinib was highly soluble in Precirol ATO5 as solid lipid and tween 80 as surfactant. Drug entrapment efficiency was found to be 83.1 % and 80.5 % for SLNs and liposomes respectively. In vitro release data revealed that 30 % of drug was released from plain suspension and more than 77 % released from both nanoformulations after 24 h at pH 7.4 respectively. By applying kinetic fit, data was most appropriately fitted into first order as compared to other. The apparent permeability of gefitinib from plain suspension, SLNs and Liposomes was found to be 3.98 × 10^?4 cm/min, 1.35 × 10^?4 cm/min and 1.79 × 10^?4 cm/min.     

A novel validated LC method for quantitation of lopinavir in bulk drug and pharmaceutical formulation in the presence of its potential impurities and degradation products

A simple isocratic liquid chromatographic method was developed for determination of lopinavir from its related impurities and assay for the first time. This method involves the use of a C(8) (Symmetry Shield RP8, 150 x 4.6 mm, 5 microm) column. The method was validated over the range of limit of quantitation (LOQ) to 120% of impurity specification limit and LOQ to 150% of working concentration for assay. The mobile phase consisted of a mixture of 50 mM of potassium phosphate buffer, acetonitrile and methanol in the ratio of 40:50:10. The flow rate was set at 1.0 mL/min with UV detection monitored at 210 nm. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. The developed method was validated for linearity, range, precision, accuracy and specificity. This method was successfully applied for content determination of lopinavir in pharmaceutical formulations. The method can be conveniently used in a quality control laboratory for routine analysis for assay and related substances as well for the evaluation of stability samples of bulk drugs and pharmaceutical formulations.     

Forced-degradation study of valdecoxib as bulk drug and in tablet formulation by HPTLC

A stability-indicating forced-degradation study of valdecoxib was conducted using high performance thin layer chromatography (HPTLC). It was used to analyze valdecoxib as bulk drug and as tablets. Undegraded valdecoxib was eluted with a retardation factor, Rf, of 0.56. Valdecoxib was forcibly degraded by exposure to alkali, acid, oxidation, and light, the greatest degradation occurring under basic conditions. Base-degraded valdecoxib gave an additional peak with an Rf value of 0.76. The calibration curve was linear in the range of 0.2-1 microg/microL with a correlation coefficient of 0.9952. Complete validation was carried out for precision (inter-day, intra-day, repeatability), accuracy, and robustness. All the data were analyzed statistically. This HPTLC procedure shows the reliability needed for use as a stability-indicating method. It can quantify valdecoxib in bulk and in tablets and also resolves the degraded peak of valdecoxib. This method is also useful for studying the degradation pattern and degradation mechanism of valdecoxib.     

Pressurised liquid extraction of flavonoids in onions. Method development and validation

A rapid and reliable analytical method for quantification of flavonoids in onions was developed and validated. Five extraction methods were tested on freeze-dried onions and subsequently high performance liquid chromatography (HPLC) with UV detection was used for quantification of seven flavonoids.The extraction efficiencies were lowest for the conventional water bath extraction compared to pressurized liquid extraction (PLE), ultrasonication, ultrasonic liquid processor, and microwave extraction, which yielded similar efficiencies. The reproducibility was in the same range (RSD: 1-11%) for all tested extraction methods. However, PLE was the preferred extraction method because the method can be highly automated, use only small amounts of solvents, provide the cleanest extracts, and allow the extraction of light and oxygen-sensitive flavonoids to be carried out in an inert atmosphere protected from light.The method parameters: extraction temperature, sample weight, flush volume and solvent type were optimised, and a clean-up step was integrated in the PLE procedure by in-cell addition of C18-material to the extraction cells, which slightly improved the recovery and reproducibility of the method. The one-step PLE method showed good selectivity, precision (RSDs = 3.1-11%) and recovery of the extractable flavonoids (98-99%). The method also appeared to be a multi-method, i.e. generally applicable to, e.g. phenolic acids in potatoes and carrots.     

Stability-indicating RP-HPLC method development and validation for determination of nine impurities in apixaban tablet dosage forms. Robustness study by quality by design approach

A quality by design (QbD) based high-resolution HPLC method is described for determination of impurities in apixaban (APX) in the tablet dosage form. Employing a simple and stability-indicating HPLC method, nine known impurities were quantified with good peak resolution. Mobile phase A (MP-A) was prepared with buffer and acetonitrile 90:10 v/v, while mobile phase B (MP-B) contained water and acetonitrile 10:90 v/v. The gradient program was 0 min, MP-A 75%, B 25%; 20 min, MP-A 65%, B 35%; 30 min, MP-A 40%, B 60%; 40min, MP-A 40%, B 60%; 42 min, MP-A 75%, B 25%; and 50 min, MP-A 75%, B 25%. The chromatographic separation was achieved using a Zorbax RX C₁₈ 250 × 4.6 mm column, 5 μm (1.0 ml min⁻¹, 280 nm, 50 μl) and a column temperature of 40°C. Several separation studies were carried out using design of experiments to optimize the method. Validation results confirm the applicability of the developed method for quality analysis and stability studies of the regular product on the manufacturing stream.     

Stability-indicating RP-HPLC method for simultaneous quantitation of tramadol and aceclofenac in presence of their major degradation products: Method development and validation

Primary objective of this study was to develop a stability-indicating reverse-phase high-performance liquid chromatography (HPLC) method for simultaneous quantitation of tramadol and aceclofenac in presence of their degradation products. The drugs were subjected to various International Conference on Harmonization recommended stress conditions, such as acid hydrolysis, alkaline hydrolysis, peroxide oxidation, thermolysis, and photolysis. The major degradation products got well resoluted from the analytes in HPLC analysis with a mobile phase composed of a mixture of 0.01?M ammonium acetate buffer (pH 6.5) and acetonitrile (65:35, v/v) through a Phenomenex Gemini C18 (250?mm?×?4.6?mm, 5?µm particle size) column. The method was linear over a range of 15–60?µg/mL for tramadol and 40–160?µg/mL for aceclofenac concentration. The analytes were detected at a wavelength of 270?nm. The method was validated and found to be specific, accurate, precise, stable, and robust for its intended use. The method can be recommended for its future use in routine quality control, accelerated and real-time stability analysis of the formulations containing tramadol and aceclofenac combination.     

Method validation for direct determination of some trace and toxic elements in soft drinks by inductively coupled plasma mass spectrometry

The consumption of soft drink beverages has increased in the last few years around the world and it is related to the diversity of brands and flavours available, increasing also the risk of ingestion of compounds considered non-beneficial to the health of consumers. In this study, fast, easy and simple method of analysis for direct determination of As, Pb, Cd, Sb, Hg, Cu, Zn, Fe, Al, Cr, Sn, Co, Mn and Ni in soft drink samples using quadrupole inductively coupled plasma mass spectrometry (Q-ICP–MS) was validated. The estimated detection limits, practical quantification limits, linearity (linear dynamic ranges and method linearity), accuracy (trueness and precision) and measurement uncertainty parameters were studied under optimised (Q-ICP–MS) conditions. The method showed that the estimated detection limits were varied between 0.02 and 2.403 µg/L, and the quantification limits were varied between 0.5 and 20 µg/L. The mean recoveries ± standard deviations at different spiking levels were varied between 75.03 ± 0.62% and 117.07 ± 2.83% and the coefficients of variation were varied between 0.49% and 9.79%. The method trueness was confirmed by using four different certified reference materials (soft drinks and treated water) purchased from FAPAS (Food Analysis Performance Assessment Scheme) and all obtained results were within satisfactory ranges and had acceptable recovery and Z-score values. The method precision, in terms of relative standard deviation, was below 4.88%. The method uncertainty expressed as expanded uncertainty of all validated elements was found to be ≤22.52%. The results obtained make the method suitable for accurate determination of validated elements in different kinds of soft drink samples at these low concentration values. Validated method was used for the determination of metallic contaminants in 40 commercial soft drink samples and the results were compared with the provisional guideline of the elements stated by Egyptian, WHO and European standards in drinking and potable bottled natural mineral water.     

Simultaneous quantification of abemaciclib and letrozole in rat plasma: method development,validation and pharmacokinetic application

Treatment through a combination of drugs involving cyclin D-dependent kinase inhibitors like abemaciclib and aromatase inhibitor like letrozole proved to be a potential therapeutic regimen and first-line treatment in estrogen receptor-positive breast cancer. In this study, we developed a simple and simultaneous RP-HPLC bioanalytical method for quantifying abemaciclib and letrozole in rat plasma. Abemaciclib and letrozole were separated on Zorbax Eclipse C₁₈ column employing a gradient elution method comprising 10 mM ammonium acetate (pH 5) and acetonitrile as mobile phase. The method was found to have acceptable selectivity, accuracy (97.20–118.17%), precision (1.10–9.39%) and stability in the validation experiment performed as per the US Food and Drug Administration guidelines. The method sensitivity was low at a concentration level of 100 ng/ml. The applicability of the method has been verified through a single-dose oral pharmacokinetic study in rat. The developed method will be useful to quantitate the analytes in rat plasma samples of different preclinical studies including their pharmacokinetic drug–drug interactions in the future. To date, no method has been reported for the quantification of abemaciclib and letrozole simultaneously in any type of biological matrices. Therefore, this study makes a definite significant contribution in the field of bioanalytical research.     

Quantitation of native and forced degraded collagens by capillary zone electrophoresis: Method development and validation

A new capillary zone electrophoresis method for collagen quantitation was developed and validated according to the International Council for Harmonization guideline Q2 (R1). The Sircol collagen assay and ultraviolet spectrometry were employed as reference methods. Capillary zone electrophoresis enables specific, simple, and fast determination within 9 min. It is less user-dependent and more automated than the Sircol collagen assay. With a limit of detection of 18.0 μg/mL, the new method is less sensitive than the Sircol collagen assay, which has a limit of detection of 6.5 μg/mL. Nonetheless, capillary zone electrophoresis covers a wider linearity range (50-400 μg/mL) compared to the Sircol collagen assay (5-80 μg/mL), with similar precision. Additional advantages of capillary zone electrophoresis are the ability to gain information on collagen integrity and to simultaneously determine native and denatured collagens. This approach represents a modern and legitimate alternative to the Sircol collagen assay. The developed method has been successfully applied to the study of three collagen products and samples from forced degradation.     

Active content determination of non-coated pharmaceutical pellets by near infrared spectroscopy: Method development, validation and reliability evaluation

A robust near infrared (NIR) method able to quantify the active content of pilot non-coated pharmaceutical pellets was developed. A protocol of calibration was followed, involving 2 operators, independent pilot batches of non-coated pharmaceutical pellets and two different NIR acquisition temperatures. Prediction models based on Partial Least Squares (PLS) regression were then carried out. Afterwards, the NIR method was fully validated for an active content ranging from 80 to 120% of the usual active content using new independent pilot batches to evaluate the adequacy of the method to its final purpose. Conventional criteria such as the R², the Root Mean Square Error of Calibration (RMSEC), the Root Mean Square Error of Prediction (RMSEP) and the number of PLS factors enabled the selection of models with good predictive potential. However, such criteria sometimes fail to choose the most fitted for purpose model. Therefore, a novel approach based on accuracy profiles of the validation results was used, providing a visual representation of the actual and future performances of the models. Following this approach, the prediction model using signal pre-treatment Multiplicative Scatter Correction (MSC) was chosen as it showed the best ability to quantify accurately the active content over the 80-120% active content range. The reliability of the NIR method was tested with new pilot batches of non-coated pharmaceutical pellets containing 90 and 110% of the usual active content, with blends of validation batches and industrial batches. All those batches were also analyzed by the HPLC reference method and relative errors were calculated: the results showed low relative errors in full accordance with the results obtained during the validation of the method, indicating the reliability of the NIR method and its interchangeability with the HPLC reference method.     

Assignment of relative configuration of desoxypropionates by 1H NMR spectroscopy: method development, proof of principle by asymmetric total synthesis of xylarinic acid A and applications

The determination of the relative configuration of 1,3-dimethyl-substituted alkyl chains is possible by interpretation of (1)H NMR shift differences. Additionally, assignments are feasible in a variety of deuterated solvents, because the corresponding shift differences are not significantly influenced by the solvent. The trends for Δδ values depending on functional groups adjacent to the stereogenic centers are shown. Based on a thorough comparison with literature data, the relative configuration of natural products can be predicted. For this purpose, we derived an empirical rule for the ranges in which Δδ values usually occur. Furthermore, we were able to proof the validity of our method by the successful prediction of the relative configuration for the polyketide natural product xylarinic acid A, which was confirmed by the asymmetric total synthesis of its enantiomer. Based on the proposed simple analysis of published (1)H NMR data and the determination of the relevant chemical-shift differences, we predicted the relative configurations of several previously unassigned natural products.     

The structural and dynamic properties of 1‐bromodecane in urea inclusion compounds investigated by solid‐state 1H, 13C and 2H NMR spectroscopy

For asymmetric guest molecules in urea, the end‐groups of two adjacent guest molecules may arrange in three different ways: head–head, head–tail and tail–tail. Solid‐state ¹H and ¹³C NMR spectroscopy is used to study the structural properties of 1‐bromodecane in urea. It is found that the end groups of the guest molecules are randomly arranged. The dynamic characteristics of 1‐bromodecane in urea inclusion compounds are probed by variable‐temperature solid‐state ²H NMR spectroscopy (line shapes, spin–spin relaxation: T₂, spin‐lattice relaxation: T_1Z and T_1Q) between 120 K and room temperature. The comparison between the simulation and experimental data shows that the dynamic properties of the guest molecules can be described in a quantitative way using a non‐degenerate three‐site jump process in the low‐temperature phase and a degenerate three‐site jump in the high‐temperature phase, in combination with the small‐angle wobbling motion. The kinetic parameters can be derived from the simulation. Copyright © 2011 John Wiley & Sons, Ltd.     

The prediction of the absolute stereochemistry of primary and secondary 1,2-diols by 1H NMR spectroscopy: principles and applications

The absolute configuration of 1,2-diols formed by a primary and a secondary (chiral) hydroxyl group can be deduced by comparison of the 1H NMR spectra of the corresponding (R)- and bis-(S)-MPA esters (MPA = methoxyphenylacetic acid). This method involves the use of the chemical shifts of substituents L1/L2 attached to the secondary (chiral) carbon, and of the hydrogen atom linked to the chiral center (C alpha-H) as diagnostic signals. Theoretical (AM1, HF, and B3 LYP calculations) and experimental data (dynamic and low-temperature NMR spectroscopy, studies on deuterated derivatives, constant coupling analysis, circular dichroism (CD) spectra, and NMR studies with a number of diols of known absolute configuration) prove that the signs of the delta delta(RS) obtained for those signals correlate with the absolute configuration of the diol. A graphical model for the reliable assignment of the absolute configuration of a 1,2-diol by comparison of the NMR spectra of its bis-(R)- and bis-(S)-MPA esters is presented.     

Analytical method development and validation of anticancer agent, 5-fluorouracil,and its metabolites in biological matrices: An updated review

Abstract

5-fluorouracil (5-FU) refers to a fluorinated pyrimidine analogue that has been widely used as an anticancer agent for colon, head, and neck cancers. Detection of 5-FU and its metabolites; 5-fluorouridine and 5-fluoro-2-deoxyuridine in biological samples allows optimization of pharmacotherapy and encourages fundamental investigations of this medication. The development of accurate and reliable sample preparation, as well as analytical methods, is critical to isolate targeted analytes from complex matrices, apart from increasing detection sensitivity of analytes. With that, this paper presents a review of prior studies pertaining to chromatographic and electrophoretic methods that focused on the analysis of 5-FU and its metabolites in biological matrices such as plasma and urine. This paper concentrates on HPLC, GC and CE systems, which are the most commonly used strategies for analytical separation of 5-FU and its metabolites from samples. Detection of these antineoplastic agents at trace level demands highly sensitive and selective analytical methodologies. Application of these analytical techniques to biological matrices is reviewed with a focus on method development strategies, including types of mobile phases and background electrolytes employed in LC and CE systems.     

De novo design of chiral organotin cancer drug candidates: validation of enantiopreferential binding to molecular target DNA and 5'-GMP by UV-visible, fluorescence, (1)H and (31)P NMR

N,N-bis[(R-/S-)-1-benzyl-2-ethoxyethane] tin (IV) complexes were synthesized by applying de novo design strategy by the condensation reaction of (R-/S-)2-amino-2-phenylethanol and dibromoethane in presence of dimethyltin dichloride and thoroughly characterized by elemental analysis, conductivity measurements, IR, ESI-MS, (1)H, (13)C and (119)Sn, multinuclear NMR spectroscopy and XRD study. Enantioselective and specific binding profile of R-enantiomer 1 in comparison to S-enantiomer 2 with ultimate molecular target CT-DNA was validated by UV-visible, fluorescence, circular dichroism, (1)H and (31)P NMR techniques. This was further corroborated well by interaction of 1 and 2 with 5'-GMP.     

青蒿素及其类似物的结构和合成 VIII:^1H核磁共振测定青蒿素降解物及有关化合物的构型

The present report deals with the stereochemistry of the degradation products of arteannuin and some intermediates of its total synthesis. The configurations of 13-CH3 and lactone ring are deduced by using spin-spin coupling constants and the pattern of 11-H as well as the NOE results from 1H NMR spectroscopy.     

Quantification of montelukast,a selective cysteinyl leukotriene receptor (CysLT1) antagonist in human plasma by liquid chromatography–mass spectrometry: validation and its application to a human pharmacokinetic study

A highly sensitive, rapid assay method has been developed and validated for the estimation of montelukast (MTK) in human plasma with liquid chromatography coupled to tandem mass spectrometry with electro spray ionization in the positive‐ion mode. Liquid–liquid extraction was used to extract MTK and amlodipine (internal standard, IS) from human plasma. Chromatographic separation was achieved with 10 mm ammonium acetate (pH 6.4): acetonitrile (15:85, v/v) at a flow rate of 0.50 mL/min on a Discovery HS C₁₈ column with a total run time of 3.5 min. The MS/MS ion transitions monitored were 586.10 → 422.10 for MTK and 409.20 → 238.30 for IS. Method validation and clinical sample analysis were performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.25 ng/mL and linearity was observed from 0.25 to 800 ng/mL. The intra‐day and inter‐day precisions were 5.97–8.33 and 7.09–10.13%, respectively. This novel method has been applied to a pharmacokinetic study of MTK in humans. Copyright © 2009 John Wiley & Sons, Ltd.