Determination Of Purity Research Articles

Method development and validation of RP-HPLC method for the determination of sumatriptan in bulk and pharmaceutical dosage form

The objective of the study was to develop RP-HPLC method for the determination of purity of sumatriptan in bulk and pharmaceutical dosage form. The development of an analytical method for the determination of drugs by HPLC has received considerable attention in recent years because of their importance in quality control of drugs and drug products. The aim of this current study was to develop a simple, rapid, precise, accurate and sensitive HPLC method for the analysis of sumatriptan in bulk and its pharmaceutical dosage form by using solvent system of TEA : ACN : methanol in the ratio 80:10:10 and C8 ODS Inertsil (250*4.6mm, 5i.d) stationary phase. The chromatographic condition is set at flow rate of 1ml/min with PDA detector at 221 nm. As per ICH requirements validation studies are carried out by using freshly prepared solutions. The linearity was demonstrated over the concentration range of 5-150 µg / ml and value was found to be as 0.99998. The %RSD of precision was found to be 0.260. The LOD and LOQ were found to be 1.967μg/ml and 5.961μg/ml respectively. Forced degradation studies were carried out under various stress conditions to demonstrate the stability-indicating capability of the developed RP-HPLC method. The proposed method was found to be simple, precise, accurate and validated according to the International Council for Harmonisation guidelines.

Validated Chiral Ultra Fast Liquid Chromatographic Method for Quantitative Analysis of Enantiomeric Vildagliptin

A rapid, accurate, and precise chiral Ultra fast liquid chromatography (UFLC) method was developed and validated for enantiomeric separation of racemic vildagliptin and S-vildagliptin according to the guidelines of the International Conference on Harmonization (ICH). The chiral chromatographic separation was achieved with a mobile phase consisting of 20 mM borax buffer (pH 9.0 ± 0.05), ACN, and 0.1% Triethylamine (50:50:0.1, v/v/v) at a flow rate of 1 ml/min using a chiralcel OD-RH column, tris(3,5-dimethyl phenyl carbamate) (250 mm × 4.6 mm, 5 μm) column. The UFLC analysis was monitored at 210 nm. The method showed good linearity with a regression coefficient (r2) of 0.999 in the range of 1 - 12 μg/ml for S-vilda. The detection limit (LOD), quantitation limit (LOQ), and the average percentage recovery for S-vilda were found to be 0.024, 0.075 μg/mL, and 99.19% to 100.4%, respectively. The percentages of relative standard deviation (% RSD) for intra- and inter-day precision were found to be 0.346% and 0.364%, respectively. The developed method proved to be reproducible as % RSD was S-vilda was found to be 99.19 w/w. The proposed chiral method can be put in application for the enantiomeric purity determination of S-vilda formulations.

A novel stability-indicating HPLC method for the determination of enantiomeric purity of eluxadoline drug: Amylose tris(3,5-dichlorophenyl carbamate) stationary phase.

A simple and sensitive stability-indicating chiral HPLC method has been developed and validated per International Conference on Harmonization guidelines for the determination of enantiomeric purity of eluxadoline (Exdl). The impact of different mobile phase compositions and chiral stationary phases on the separation of Exdl enantiomer along with process- and degradation-related impurities has been studied. Homogeneity of Exdl and stable results of Exdl enantiomer in all degraded samples reveal the fact that the proposed method was specific (stability indicating). Amylose tris(3,5-dichlorophenyl carbamate) stationary phase column Chiralpak IE-3 (150 ×4.6mm, 3μm) provided better resolution with polar organic solvents than cellulose derivative, crown ether, and zwitterion stationary phases and nonpolar solvents. The mobile phase consisted of acetonitrile, tetrahydrofuran, methanol, butylamine, and acetic acid in the ratio of 500:500:20:2:1.5 (v/v/v/v/v). Isocratic elution was performed at a flow rate of 1.0mL/min, column temperature of 35°C, injection volume of 10μL, and UV detection of 240 nm. The United States Pharmacopeia (USP) resolution of the Exdl enantiomer was found to be more than 4.0 within a 65-min run time. Exdl enantiomer detector response linearity over the concentration range of 0.859-4.524 μg/mL was found to be R2 =0.9985. The limit of detection, limit of quantification, and average percentage recovery values were established as 0.283 μg/mL, 0.859 μg/mL, and 96.0, respectively.

Determination of purity and anionic exchange efficiency of amino acid ionic liquids synthesis by multiple-injection capillary zone electrophoresis

The purity of ionic liquids (IL) is important for its performance, so the methods able to quantify low concentration impurities are necessaries. Therefore, this paper aims to determine the purity and the anionic exchange efficiency for the preparation of amino acids ionic liquids (AAIL). For this, a Multiple-Injection Capillary Zone Electrophoresis (MICZE) method with direct UV detection was developed and optimized for iodide (I−) determination as impurity of AAIL synthesis, comparing this method with traditional injection modes. Furthermore, this paper aims to demonstrate the use of factorial design methods for the optimization of MICZE method. The method optimization allowed five consecutives I− injections of sample in a single run, with analysis time of less than 3 min, showing a larger analytical frequency, counting with 31 injections per hour. It was possible to determine the high purity of the prepared and analyzed AAIL, ranging from 89% to 95% and its overall ionic exchange yield varying from 55% to 87%.

Differential NMR and chromatography for the detection and analysis of adulteration of vetiver essential oils

Adulteration of essential oils is a common and serious problem. Adequate and fast methods are required to establish the authenticity and purity. GC-MS, 1H and 13C NMR were compared in combination with similarity calculations as well as differential spectroscopy and chromatography for the authentication and determination of purity of vetiver essential oils. In the investigation of eight commercial oils 1H and 13C NMR adequately detected all six adulterants encountered in four of the commercial samples, while GC-MS was not able to detect adulteration with vegetable oils. A great advantage of the combined use of similarity calculation and NMR is that the authenticity can be verified without the need of concomitant measurement of a standard sample. The calculation can be carried out with a registered reference spectrum, in the case of 1H NMR acquired using the same magnetic field, but in the case of 13C NMR the spectrum can be obtained with another magnetic field. 1H NMR has the advantage of high speed, and the results can be obtained within minutes. 13C NMR was found to be superior in its ability to provide unequivocal identification of eventual adulterants, with differential spectroscopy revealing all signals of the adulterants.

Selection of breed-specific SNPs in three Indian sheep breeds using ovine 50 K array

Recently DNA-based genetic purity determination tools have gained popularity, especially, in small ruminants like sheep. The objective of the present study was to select breed-specific SNPs for the three commercially important sheep breeds of India, i.e., Changthangi, Deccani, and Garole. Sheep genotype data (Ovine 50 K SNP array) from 344 individuals of eight sheep breeds (Garole, Deccani, Changthangi, Tibetan, Australian Merino, Dorset Horn, Rambouillet, and Irish Suffolk) were used as a reference population. Here, we used pre-selection statistics and the minor allele frequency–linkage disequilibrium (MAF-LD) method to analyze the reference population dataset. SNPs selection and breed assignment was executed with a panel of 768 markers for eight breeds near intermediate gene frequencies. We identified 95, 89, and 92 breed-specific SNPs for Changthangi, Deccani, and Garole, respectively. These informative SNPs were found to be associated with essential candidate genes associated with production and disease resistance. This methodology may be implemented in practice to identify individuals as being purebred or not.

Comparison of the Properties of Cellulose Obtained from Various Sources

The article discusses the structure, structure and properties of cellulose obtained from various natural sources of plant origin. The physical and mechanical properties of celluloses (content of α–cellulose, determination of the mass fraction of pentosans; determination of the copper number; viscosity characteristics; determination of the degree of purity, moisture content and ash content and determination of the average molecular weight, degree of polymerization and content of aldehyde and carboxyl groups), interaction with nitrogen acid as well as the ability of various cellulose samples to complex formation with copper ions.

Quality assessment of five pharmaceutical products of different brands marketed in Nigeria

Many pharmaceutical products have been reported to be substandard and counterfeit. About 15% of all drugs circulating in some parts of Africa and Asia are believed to be counterfeit, with figures rising to as high as 50%. This study was therefore carried out to assess the quality of five groups of drugs of different brands manufactured by pharmaceutical companies in Nigeria. Qualitative and quantitative analysis were carried out on the drugs using United States, British and Indian Pharmacopoeias. Qualitative analysis investigated includes: friability test, hardness test, disintegration test, dissolution test, pH, and weight uniformity for tablets; specific gravity, flow rate, pH and weight per ml of syrups. The quantitative analysis includes percentage purity and drug assay determination for different constituents using titrimetric, spectrophotometric and high performance liquid chromatographic (HPLC) methods. From the study, it was established that the Nigeria brands of pharmaceutical products sampled generally conformed to the established qualitative and quantitative specifications. There was however a short slight fall in the chromatographic assay for vitamin B6 in the Blood tonic from one company and similarly, the vitamin B1 in the Blood tonic of one company were at variance to those from other companies. The result for the statistical analysis shows that there is uniformity in weight of all tablets analysed and it complies with the specification. Standard pharmaceutical analysis must therefore be regularly carried out on pharmaceutical products before marketing so as to ensure good health, safety, elimination of sub-standard drugs and prevention of over- or under-dosage, which may further affect or cause damage to the health of the users. Consumer’s choice of generics should therefore be based on the analytical results (assay results, bio-pharmaceutical results, and statistical results) of the product and the projected bioequivalence.

Elaboration and Validation of HPLC/DAD Method for Quality Control of Products Containing Cannabidiol

Background: Cannabidiol (CBD) is the main, physiologically active, but psycho-inactive constituent of the glandular hairs of Cannabis sativa. CBD exhibits diverse pharmacological activities and it is used as an anticonvulsant, sedative and hypnotic agent. Other biological properties shown by CBD are antipsychotic, anti-inflammatory and neuroprotective. Objective: The aim of this study is to develop, validate and apply high performance liquid chromatography (HPLC/DAD) method with diode array detection for identification and assay of CBD in food supplements obtained by different types of extraction and purification. Method: HPLC method with isocratic elution using column C18 ODS, 250 x 4.6, 100 Å, 5 μm, mobile phase - a mixture of acetonitrile and water in ratio 80:20 v/v, a flow rate of 1.0 ml/min and DAD detection at 220 nm, was used. Results: The method was developed and validated according to European Pharmacopoeia and International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). For the analytical purposes, parameters like repeatability, linearity, accuracy, LOD, LOQ, specificity and system suitability were studied. The developed method was successfully applied on a series of food supplements containing CBD. Conclusion: The developed method is of practical importance for the determination of purity, identification and assay tests of food supplements containing CBD extract in accordance with EU regulation concerning food authorizations because it is useful for analytical tests for identification, purity determination and assay of products containing CBD extract and could serve as part of the testing process in the pharmaceutical industry.

Single radio UHPLC analysis for the quality control of technetium-99m radiolabelled radiopharmaceuticals

The radiochemical purity (RCP) determination of radiopharmaceuticals is routinely done with radio-thin layer chromatography (r-TLC). These methods are usually transposed and adjusted from the summary product characteristics without any analytical validation. The r-TLC method is simple but manually-performed steps could lead to RCP misinterpretation. To increase the sensitivity, radio ultra-high performance liquid chromatography (r-UHPLC) can be used. In this study, an r-UHPLC method had been validated and compared to the r-TLC method. Hydrolyzed-reduced technetium had also been studied.

Synthesis and Characterization of Optically Pure Gamma‐PNA Backbones by SIBX‐Mediated Reductive Amination

Chiral peptide nucleic acid (PNA) is a derivative of regular PNA by introducing a chiral center to its backbone, and is known to bind more strongly to DNA or RNA than regular PNA. In particular, in the case of a γ‐backbone, the L isomer stabilizes the PNA/DNA duplex, and the D‐isomer has the opposite effect. Therefore, the synthesis of an optically pure γ‐backbone is very important. Here, we report a novel synthetic strategy for the suppression of epimerization during the synthesis of the γ‐PNA backbone. A stabilized form of 2‐iodoxybenzoic acid (SIBX) was used as an oxidative reagent in the key intermediate of the N‐Boc‐amino acetaldehyde synthesis. This paper reports (1) the synthesis and comparison of three different γ‐PNA backbones (lysine, alanine, and glutamate) by three different synthetic routes (SIBX, lithium aluminum hydride, and Red‐Al) and (2) the determination of chiral purity from their derivative compounds. The enantiomeric excess purity of SIBX‐mediated γ‐PNA backbones was determined to be more than 99.4%, as ascertained by the high‐performance liquid chromatography (HPLC) chromatogram on a standard RP‐C18 column. It is comparatively higher than that of the other methods examined in this work.

Development of pharmacognostic parameters for the leaf of Bridelia scandens (Roxb.) Willd

Background: Pharmacognostic study of medicinal plants is an important parameter for standardization and authentication of plants, with the help of which adulteration and substitution can be prevented. The present study deals with pharmacognostic profile of leaf of Bridelia scandens (Roxb).Willd. an important traditional plant, belonging to family Euphorbiaceae used to treat various ailments. Methods: The present study includes macroscopic and microscopic studies, quantitative microscopy, and physiochemical characters such as ash value, extractive values, fluorescence analysis, and total phenol and flavonoid content. Results: Macroscopically, the leaves are B. scandens are elliptic –oblong or obavate, dark green above, pale green below lateral veins. Microscopically, leaf consists of thick semicircular midrib and the lamina, cortical zone ending with thick continuous cylinder of sclerenchyma cells. Sclerenchyma cylinder completely enclosed the vascular cylinder of the midrib, consists of only continuous thick layer of phloem. Xylem cylinder consists of numerous short or long radial chains of vessels. The lateral vein is flat on the adaxial side and prominently projecting hemispherical body on the adaxial side. Powder microscopy of leaf revealed the presence of spiral xylem vessels, rosette and prismatic calcium oxalate crystals and trichomes. A Physiochemical characteristic was also determined. Conclusion: Existing literature revealed that so far, no Pharmacognostic study has been reported on the leaf of B. scandens. Findings from this investigation can be used for its identification and determination of quality and purity of medicinally important plant. Thus exploring the usefulness of pharmacognostic evaluation to validate and authenticate drug.

Purity Determination of Cyclophosphamide Hydrate by Quantitative <sup>31</sup>P-NMR and Method Validation

Recently, quantitative NMR (qNMR), especially 1H-qNMR, has been widely used to determine the absolute quantitative value of organic molecules. We previously reported an optimal and reproducible sample preparation method for 1H-qNMR. In the present study, we focused on a 31P-qNMR absolute determination method. An organophosphorus compound, cyclophosphamide hydrate (CP), listed in the Japanese Pharmacopeia 17th edition was selected as the target compound, and the 31P-qNMR and 1H-qNMR results were compared under three conditions with potassium dihydrogen phosphate (KH2PO4) or O-phosphorylethanolamine (PEA) as the reference standard for 31P-qNMR and DSS-d6 as the standard for 1H-qNMR. Condition 1: separate sample containing CP and KH2PO4 for 31P-qNMR or CP and DSS-d6 for 1H-qNMR. Condition 2: mixed sample containing CP, DSS-d6, and KH2PO4. Condition 3: mixed sample containing CP, DSS-d6, and PEA. As conditions 1 and 3 provided good results, validation studies at multiple laboratories were further conducted. The purities of CP determined under condition 1 by 1H-qNMR at 11 laboratories and 31P-qNMR at 10 laboratories were 99.76±0.43% and 99.75±0.53%, respectively, and those determined under condition 3 at five laboratories were 99.66±0.08% and 99.61±0.53%, respectively. These data suggested that the CP purities determined by 31P-qNMR are in good agreement with those determined by the established 1H-qNMR method. Since the 31P-qNMR signals are less complicated than the 1H-qNMR signals, 31P-qNMR would be useful for the absolute quantification of compounds that do not have a simple and separate 1H-qNMR signal, such as a singlet or doublet, although further investigation with other compounds is needed.

Screening of 200 Core SNPs and the Construction of a Systematic SNP-DNA Standard Fingerprint Database with More Than 20,000 Maize Varieties

To strengthen the management of maize varieties and the protection of intellectual property rights to new varieties, we constructed a comprehensive single nucleotide polymorphism (SNP)-DNA standard fingerprint database of 20,075 materials covering nationally and provincially approved maize hybrid lines, hybridized combinations, and inbred lines. The database was based on 200 core SNPs selected from 60 K SNPs distributed in intragenic regions, including 106 (53.0%) located in exons. Average minor allele frequencies (MAF) of the 200 SNPs in 6755 maize hybrids, 7837 hybridized combinations, and 3478 inbred lines were 0.385, 0.350, and 0.378, respectively, with corresponding average polymorphism information content (PIC) values of 0.354, 0.335, and 0.351. Heterozygous genotype frequencies of maize hybrids, hybridized combinations, and inbred lines averaged 0.48, 0.47, and 0.012, respectively. The number of different loci in the three different maize groups ranged from one up to 164, 160, and 140, respectively. The percentage of different SNPs within 5% (the number of difference SNPs is less than 10) accounted for 0.013%, 0.011%, and 0.030% among pairwise comparisons of samples within hybrid lines, hybridized combinations and inbred lines, respectively. Genetic distances between varieties based on the 200 core SNPs were highly correlated with those obtained using 60 K SNPs, with a correlation coefficient of 0.82 and 0.87 in in inbred and hybrid lines, respectively. The maize SNP-DNA fingerprint database established in this study can play an important role in variety authentication, purity determination and the protection of variety rights, thereby providing reliable, comprehensive data support for use in the seed industry.

Rapid Estimation of T1 for Quantitative NMR.

Quantitative NMR spectroscopy (qNMR) is an essential tool in organic chemistry, with applications including reaction monitoring, mechanistic analysis, and purity determination. Establishing the correct acquisition rate for consecutive qNMR scans requires knowledge of the longitudinal relaxation time constants (T1) for all of the nuclei being monitored. We report a simple method that is about 10-fold faster than the conventional inversion recovery technique for the estimation of T1.

Development of a chiral HPLC method for the separation and quantification of hydroxychloroquine enantiomers

Hydroxychloroquine (2-[[4-[(7-Chloroquinolin-4-yl) amino]pentyl](ethyl) amino]-ethanol, HCQ), an effective anti-malarial drug, has been tested in the clinics for potential treatment of severe coronavirus disease 2019 (COVID-19). Despite the controversy around the clinical benefits of HCQ, the existence of a chiral center in the molecule to possess two optical isomers suggests that there might be an enantiomeric difference on the treatment of COVID-19. Due to their poor resolution and the inability of quantification by previously reported methods for the analysis of HCQ enantiomers, it is necessary to develop an analytical method to achieve baseline separation for quantitative and accurate determination of the enantiomeric purity in order to compare the efficacy and toxicity profiles of different enantiomers. In this study, we developed and validated an accurate and reproducible normal phase chiral high-performance liquid chromatography (HPLC) method for the analysis of two enantiomers of HCQ, and the method was further evaluated with biological samples. With this newly developed method, the relative standard deviations of all analytes were lower than 5%, and the limits of quantification were 0.27 μg/ml, 0.34 μg/ml and 0.20 μg/ml for racemate, R- and S-enantiomer, respectively. The present method provides an essential analytical tool for preclinical and clinical evaluation of HCQ enantiomers for potential treatment of COVID-19.

Purity determination and uncertainty estimation of natural products sourcing chemical reference substances by high-performance liquid chromatography and differential scanning calorimetry

Natural products sourcing chemical reference substances (NPSCRSs) are widely used in the analysis industry to determine the content and quality of food and drug products. Chromatographic techniques have been traditionally used to determine the purity of chemical reference substances (CRSs). However, the uncertainty values of NPSCRSs purity are rarely measured due to the chemical complexity of natural products and the diversity of instrument influencing factors. Recently, differential scanning calorimetry (DSC) technique has been proved to be able to detect the purity of CRSs, but its adaptability for NPSCRSs is rarely researched. Herein, a HPLC-DSC combined method was developed for the purity calibration and uncertainty measurement of 20 NPSCRS samples. As a result, the value of “purity ± uncertainty” was suggested to represent the quantitative result for purity measurement of NPSCRSs. Among four influence factors of HPLC analysis, the influence of peak area was first proposed as the source of uncertainty, and the uncertainty caused by the wavelength was simplified to a five wavelength-based method. As for DSC method using for purity determination of NPSCRSs, it could be used as an alternative and complementary method to verify the purity of a NPSCRS that is more than 98% because of its advantage of practicality, speed, reproducibility, no corresponding reference standard requirement, and no sample preparation is required. Note that, DSC method is not appropriate if the compound lacks standard melting peak, decomposes in the defined temperature range, or exhibits other thermal event (s) which interfere with the melting point of the compound.

A Step Towards Optimization of the qNMR Workflow: Proficiency Testing Exercise at an GxP-Accredited Laboratory

Quantitative nuclear magnetic resonance (qNMR) is considered as a powerful tool for multicomponent mixture analysis as well as for the purity determination of single compounds. Special attention is currently paid to the training of operators and study directors involved in qNMR testing. To assure that only qualified personnel are used for sample preparation at our GxP-accredited laboratory, weighing test was proposed. Sixteen participants performed six-fold weighing of the binary mixture of dibutylated hydroxytoluene (BHT) and 1,2,4,5-tetrachloro-3-nitrobenzene (TCNB). To evaluate the quality of data analysis, all spectra were evaluated manually by a qNMR expert and using in-house developed automated routine. The results revealed that mean values are comparable and both evaluation approaches are free of systematic error. However, automated evaluation resulted in an approximately 20% increase in precision. The same findings were revealed for qNMR analysis of 32 compounds used in pharmaceutical industry. Weighing test by six-fold determination in binary mixtures and automated qNMR methodology can be recommended as efficient tools for evaluating staff proficiency. The automated qNMR method significantly increases throughput and precision of qNMR for routine measurements and extends application scope of qNMR.

Determination of biodiesel purity through feature mapping to the multi-dimensional space by the LS-SVM approach

Purity is one of the essential properties of biodiesel. Since the purity parameter depends on different operating conditions, its direct measurement is too hard and can only be obtained for specific ranges of conditions. Therefore, this work considers the least-squares support vector machines (LS-SVMs) that transform operating conditions to a multi-dimensional space to simulate biodiesel purity in wide ranges of operating conditions. Indeed, we develop a reliable LS-SVM approach for modeling the biodiesel purity as a function of catalyst type and its concentration, reaction time, temperature, methanol-to-oil volume ratio, frequency, and amplitude of ultrasonic waves. The designed LS-SVM’s predictive performance is compared with four available artificial intelligence (AI) techniques in reliable literature. The obtained results confirm that the LS-SVM paradigm outperforms other considered AI-based techniques regarding five different statistical criteria. Our LS-SVM model provides AARD = 2.2%, RMSE = 3.46, and R2 = 0.9868 for the prediction of 267 experimental data points, which includes 267 data points. This model is finally employed for investigating the effect of different influential variables on biodiesel purity.

Validation of an analytical method for the quantification of human fibrinogen in pharmaceutical products by size-exclusion liquid chromatography (SEC-HPLC).

Fibrinogen plays a vital role in normal homeostasis by promoting platelet aggregation, clot formation and fibrinolysis. It is quantified in finished pharmaceutical products using different methods described in pharmacopoeia, but these are inaccurate, difficult to validate and do not allow for identification of aggregates or protein products of the same formulation. The aim of this study was to develop and validate a method for quantification of the content of fibrinogen and other proteins present in pharmaceutical formulations by comparing it with current pharmacopeial methods. Fibrinogen was quantified in two commercial products and compared to a pharmacopeial method using a validated method for size-exclusion high-pressure liquid chromatography (SEC-HPLC). The fibrinogen level was in accordance with both products' specifications. The SEC-HPLC method showed that the percentage of fibrinogen was 94.88 for one product and 50.68 for the other, and detected high molecular weight aggregates in the second product. The SEC-HPLC method that we developed is an improvement to the current pharmacopeial method, because it allows for quantification of fibrinogen and determination of product purity. This is important because greater purity can reduce potential adverse effects of pharmaceutical products in patients.