Peak Purity Analysis Research Articles

AQbD-guided development and validation of an innovative extraction procedure and stability-indicating RP-HPLC method for quantification of posaconazole in tablet formulation

The objective of this work is to develop a stability-indicating HPLC method for the quantification of Posaconazole (PCZ) in tablet formulation using an Analytical Quality by Design (AQbD) approach. The development process involved the Design of Experiments (DOE) utilizing distinctive constraints mixture design for mobile phase ratio optimization and a 2-level factorial design for selection of extraction diluent compositions. Key responses measured included % assay and system suitability parameters. Method operable design regions (MODR) were determined, and final optimum conditions were selected. Forced degradation studies were conducted to assess method stability. The optimized HPLC method employed a Zorbax C18 column with a mobile phase consisting of pH 3.5 10mM phosphate buffer, acetonitrile, and methanol in a ratio of 30:53:17 % v/v/v. The method demonstrated stability-indicating capabilities, with PCZ degradation observed in acidic and oxidative environments, while remaining stable in alkali. Peak purity analysis from Empower software confirmed the absence of interaction with degradants. Validation according to ICH Q2 (R2) guidelines showed precision, linearity over the range of 0.25 µg/mL to 376 µg/mL, and accuracy demonstrated through recovery studies from 50 to 150%. The developed HPLC method utilizing AQbD approach is specific, robust, precise, and accurate for the quantification of PCZ in tablet formulations, thus suitable for routine analysis.

Estimation of Lexisinatide in Bulk and Tablets by RP‐UPLC Method Developed by Quality by Design Concept

AbstractThe primary goal of the proposed study is to establish and validate a low‐cost, sensitive, reliable, and trouble‐free RP‐UPLC process with improved performance for the direct estimation of Lixisenatide in pure powder and fixed‐dose tablet form. The proposed method is optimized by implementing quality by design studies. The method is optimized using quadratic model of central composite design (CCD) of randomized response surface study. Successful separation of Lixisenatide is obtained by the use of Phenyl‐XBD (1.7 µm, 2.1 mm × 100 mm) column and a mobile phase of acetonitrile: 0.1% w/v OPA (60:40) with 0.5 mL min−1 flow rate. The separated Lixisenatide and its degradants are explorated with PDA detector which has the ability to provide comprehensive spectral information, rapid data acquisition, quantitative analysis, and peak purity analysis of all compounds simultaneously. The RT of Lixisenatide is found to be 1.34 min. The lowest concentration of Lixisenatide's limit of detection and limit of quantification makes guarantees regarding the method's sensitivity. For the provided range of linear concentrations (1.25–7.5 µg mL−1), the regression coefficient is found to be 0.999. The computed average percentage recoveries of Lixisenatide in spiked solutions ranged from 98.9% to 101.2%. The % degradation of Lixisenatide in the presence of a variety of forced conditions confirms the existing method's stability. Lixisenatide is significantly photo sensitive when compared to other stressful conditions such as acid, base hydrolysis, and oxidation. The proposed UPLC approach separates Lixisenatide with high sensitivity, a short retention time, and an affordable solvent system.

Novel stability indicating HPLC method for the quantification of Nirmatrelvir in bulk drugs

Nirmatrelviris anantiviral drug that acts asa 3C-likeprotease inhibitor. It is used with the combination of Ritonavir for the treatment of COVID-19. The World Health Organization strongly recommended this medicine to moderate and mild COVID patients, which is now the best option for COVID-19 therapeutics against SARS-CoV-2. As a result, there is an urgent need for quick and low-level quantitative analytical techniques for Nirmatrelvir and its products by focusing on the mass-compatible buffer with a shorter run time as the target. Accordingly, we developed a procedure with the following optimized conditions; mobile phase: 0.10 % formic acid in water and acetonitrile (55:45 %v/v), column oven temperature: 45 °C, flow rate: 1.0 mL/min with 15 min run time in isocratic elution mode. The developed procedure is linear and accurate from 0.20 to 500 µg/mL. Forced degradation studies in all solid and liquid degradations were executed to demonstrate the method's stability indicative capacity. To demonstrate the peak purity of the desired compound orthogonally, all degradation samples were then subjected to peak purity analysis using an HPLC PDA (High-performance liquid chromatography photodiode array) detector and LCMS (Liquid chromatography-mass spectrometry) analysis. The literature methods were developed with highly concentrated buffers which cause ion suppression in LCMS and increase backpressure in HPLC systems but the proposed method is conducive and more sensitive (limit of quantitation: 0.20 µg/mL and limitation of detection: 0.0 6 µg/mL) than the reported methods and has a wide linearity range as well. The developed technique has been fully validated by ICH (International Council for Harmonisation) guidelines. Further, it has been evaluated for greenness character using the AGREE (Analytical GREEnness) statistical tool and the obtained score is 0.61 out of 1.

A Standardized 2D-LC Screening Platform for Peak Purity Determination in Pharmaceutical Analysis

The determination of peak purity in chromatographic methods used in pharmaceutical analysis is a vital step in evaluating specificity to ensure the safe production of medicines. Current peak purity assessments accomplished through diode array detectors (DAD) and mass spectrometry (MS) have some challenges that could lead to misinterpreted purity values when the sample under evaluation is susceptible to the limitations of these techniques. Two-dimensional liquid chromatography (2D-LC) has the ability to resolve difficult-to-separate mixtures, and has been widely adapted to biological compounds. This study proposes a 2D-LC screening method as a means of determining peak purity of active pharmaceutical ingredients (API) and their related substances. Results confirm that the 2D-LC screen was successful in separating API/impurity mixtures in all 10 test cases studied. Selection of screening conditions including mobile phase choice and column selection is discussed along with the effectiveness and idiosyncrasies of peak purity analysis by 2D-LC.

Development of Stability Indicating and Robust RP-HPLC Method for Determination of Teneligliptin

A simple and rapid reverse-phase HPLC method was developed for determination of Teneligliptin (TGP) in the presence of its degradation products generated from forced decomposition studies. The HPLC separation was achieved on a C18 ACE column (150x 4.6 mm i.d.; 5 μm) using mobile phase as a mixture of Phosphate buffer pH-7.2 using ortho-phosphoric acid: methanol (30:70v/v). The UV detection was carried out at 245nm at ambient temperature and the flow rate of 1.0 mL/min. The calibration curve was found to be linear in the concentration range of 10-50 μg/mL(r=0.9993). Force degradation study was performed under various conditions like acidic, alkaline, oxidative, photolytic and mass balance calculations were carried out from the degradation results. The developed method was validated as per ICH guidelines with respect to linearity, accuracy, precision, limit of detection and quantification. The robustness of the proposed method was evaluated by the Plackett Burman design. The purity of the degraded sample was checked by peak purity analysis. The peaks of degradation products did not interfere with that of pure Teneligliptin.

Improving visualization of trace components for quantification using a power law based integration approach

In some cases, trace component analysis only requires a sensitive and high-resolution mass spectrometer. However, enantiomers must be completely separated to be differentiated with a mass spectrometer, which is highly dependent on the stationary-mobile phase composition. In case of a challenging chiral separation, instead of trying new columns for screening purpose, resolution enhancement techniques could be used to resolve partially overlapping peaks. A well-known enhancement method is the power law, which increases the linear dynamic range of each analyte and reduces excessive noise. In many cases, the peak noise can decrease significantly by applying the power law. However, the main drawback is that this approach changes relative peak areas and heights of each peak in a non-linear fashion which limits its use for quantitative purposes. In this study, a normalized power law was utilized for extracting correct area information. It is a simple (5 step) protocol that only requires Microsoft Excel, and results in enhanced visualization of trace components, especially in low signal/noise environments, and makes integration convenient and reproducible. Several difficult chiral trace component analyses were investigated, including applications pertaining to ultrafast high-throughput chromatography, enantiopurity, and peak purity analysis. For complicated cases with multiple overlapped peaks of different resolutions, a segmented normalized power law was utilized. A trace component coeluting near a dead volume peak and a trace enantiomeric component in the tail of the corresponding enantiomeric peak were virtually enhanced. As an additional tool, first and second derivatives were utilized to identify if an enantiomeric impurity is coeluting with the dominant enantiomer under overload conditions. Idiosyncrasies of the derivative test are discussed. This study shows how these simple approaches can be used for accurate quantitation, specifically for trace enantiomeric components.

Validated reverse phase HPLC diode array method for the quantification of intact bevacizumab, infliximab and trastuzumab for long-term stability study

The aim of the present study was to develop suitable and reliable method for quantification three of the most worldwide used therapeutic monoclonal antibodies (mAbs) -bevazizumab (BVZ), infliximab (INF) and trastuzumab (TTZ)- to be used in long-term stability studies. Reverse phase (RP) was selected by its greater sensibility and reproducibility comparing with other chromatographic modes. Then a high performance liquid chromatography with diode array detection (RP)HPLC/DAD method was checked. Since the three mAbs represent the active ingredient in the medicines in which they are formulated, the selected method was validated for each one in accordance with the International Conference on Harmonization (ICH) guidelines for pharmaceuticals for human use. Then method was validated in terms of linearity, accuracy, precision, (repeatability, intermediate precision) specificity (by forced degradation studies), robustness and system suitability. Spectral peak purity analysis strategy was used to test mAb degradations. Comparative study of the results indicated similar behavior for the three mAbs. Forced degradation studies also provided deep knowledge of these important bio-macromolecules. At last the method was successfully used to quantify BVZ, INF and TTZ in long-term stability studies performed under hospital conditions of use and they showed great stability regarding quantification during the time of the study.

Influence of different stabilizers on the encapsulation of desmopressin acetate into PLGA nanoparticles

To address targeting and bioavailability issues of peptidic drugs like desmopressin, the encapsulation into nanoparticles (NP) has become standard in pharmaceutics. This study investigated the encapsulation of desmopressin into PLGA NP by the use of pharmaceutically common stabilizers as a precursor to future, optional targeting and bioavailability experiments. Polymer dry weights were measured by freeze drying and thermo gravimetric analysis (TGA). Particle sizes (ranging between 105 and 130nm, PDI<0.1) and zeta potentials (−35 to −45mV) were analyzed with Dynamic Light Scattering (DLS) and Laser-Doppler-Anemometry (LDA) respectively. Highest loading efficiencies, quantified by RP-HPLC, were achieved with Pluronic F-68 as stabilizer of the inner aqueous phase (1.16±0.07μg desmopressin/mg PLGA) and were significantly higher than coating approaches and approaches without stabilizer (0.74±0.01μg/mg). Optimized nanoformulations are thus in competition with the concentration of commercial non-nanoparticulate desmopressin products. Stability of desmopressin after the process was evaluated by HPLC peak purity analysis (diode array detector) and by mass spectrometry. Desmopressin was shown to remain intact during the whole process; however, despite these very good results the encapsulation efficiency turned out to be a bottle neck and makes the system a challenge for potential applications.

A stability indicating simultaneous dual wavelength UV–HPLC method for the determination of potential impurities in fampridine active pharmaceutical ingredient

A novel, sensitive, stability indicating simultaneous dual wavelength reverse phase UV–HPLC method has been developed for the quantitative determination of potential impurities of fampridine active pharmaceutical ingredient. Efficient chromatographic separation was achieved on a C18 stationary phase in gradient mode and quantitation by ultraviolet dual wavelength detection. The method was validated according to ICH guidelines with respect to specificity, precision, linearity and accuracy. Regression analysis showed correlation coefficient value greater than 0.999 for fampridine and its seven impurities. Detection limit as low as 0.003% was achieved for fampridine N-oxide and 0.01% for other impurities. Accuracy of the method was established based on the recovery obtained between 93.3% and 110.0% for all impurities. The method was found to be specific, selective to the degradation products and robust. Peak purity analysis by PDA detector confirmed the specificity of the method. Major degradation of the drug substance was found to occur under oxidative stress conditions to form fampridine N-oxide.

HPLC-DAD/ESI-MS identification and quantification of polar constituents in six Stachys taxa from Balkan peninsula

The genus Stachys L. comprises over 450 species [1] and is one of the largest genera of the Lamiaceae. Aerial parts of Stachys spp. are used traditionally to treat genital tumors, sclerosis of the spleen, inflammatory diseases, cough and ulcers [2]. In previous study an HPLC-DAD-MS method was developed for the quality control of S. recta [3]. Aim of the present work was to develop an HPLC-DAD/ESI-MS method which may serve as a tool for future chemotaxonomic and quality control studies of Stachys spp. Six mostly endemic Stachys taxa from Balkan peninsula were studied: S. anisochila Vis. & Pančic, S. alpina L. subsp. dinarica Murb., S. atherocalyx K. Koch, S. beckeana Dörfl. & Hayek, S. nitens Janka and S. plumosa Griseb. A RP-C18 solvent saving column was chosen and the developed assay permitted a good separation among different constituents of the extracts. Overall, 42 constituents were detected and identified, belonging to three classes of secondary metabolites: flavone glycosides (isoscutellarein and hypolaetin derivatives), phenylethanol glycosides and caffeic acid derivatives. S. plumosa instead, contained chrysoeriol/apigenin derivatives. Peak purity analysis and peak assignment were accomplished by means of MS and reference compounds. The analytical method provided a good separation of the constituents, concerning both marker constituents (flavonoids) and the phenylethanol glycosides. Quantification results showed that these plants are a good source of flavonoids. All validation criteria (linearity, LOD, LOQ, intra- and inter-day precision, time precision, accuracy) were fulfilled. This method is a reliable and accurate tool for the detailed analysis of Stachys taxa.

Perspectives on Method Validation: Validation is a Multistep Process with USP Regulatory Guidelines at each Step

<b>Perspectives on Method Validation</b>: Validation is a Multistep Process with USP Regulatory Guidelines at each Step

Peak purity determination with principal component analysis of high-performance liquid chromatography–diode array detection data

A method is proposed for the determination of chromatographic peak purity by means of principal component analysis (PCA) of high-performance liquid chromatography with diode array detection (HPLC–DAD) data. The method is exemplified with analysis of binary mixtures of lidocaine and prilocaine with different levels of separation. Lidocaine and prilocaine have very similar spectra and the chromatograms used had substantial peak overlap. The samples analysed contained a constant amount of lidocaine and a minor amount of prilocaine (0.02–2 conc.%) and hence the focus was on determining the purity of the lidocaine peak in the presence of much smaller levels of prilocaine. The peak purity determination was made by examination of relative observation residuals, scores and loadings from the PCA decomposition of DAD data over a chromatographic peak. As a reference method, the functions for peak purity analysis in the chromatographic data system used (Chromeleon) were applied. The PCA method showed good results at the same level as the detection limit of baseline-separated prilocaine, outperforming the methods in Chromeleon by a factor of ten. There is a discussion of the interpretation of the result, with some comparisons with evolving factor analysis (EFA). The main advantage of the PCA method for determination of peak purity over methods like EFA lies in its simplicity, short time of calculation and ease of use.

Peak Purity Determination with a Diode Array Detector#

Peak purity analysis is an evaluation technique for detecting the presence of coeluting impurities in high performance liquid chromatography (HPLC) data. Running a peak purity check prior to analytical quantitation helps to ensure accuracy. In the development of analytical methods, peak purity analysis can reveal the presence of contamination during standardization and, by doing so, prevent the subsequent generation of false analytical data. Peak purity analysis is also a useful addition to routine quality control procedures, especially in the analysis of pharmaceuticals and food products, for which contamination and quality of results are critical. #Reprinted from the Encyclopedia of Chromatography (© 2003); URL: http://www.dekker.com/servlet/product/productid/E‐ECHR.

New methods for spectrometric peak purity analysis in chromatography

Simple methods for peak purity analysis in chromatography are proposed for accurately assaying the chromatographic or electrophoretic process by spectroscopy. With these methods, the purity at each point of a chromatographic peak containing an unknown type and number of components can be detected and visualized. The `absorbance (A) diagram' is constructed by plotting the absorbance values of different wavelengths against each other. When only straight lines are obtained for different wavelength combinations, only one component was detected in the corresponding peak. Bent curves in the A diagrams mean that at least two components were registered. Straight lines in the absorbance difference quotient (ADQ) diagrams indicate that two components occur in the corresponding peak. Bent curves signify that at least three components were registered. The new methods are demonstrated using the HPLC technique for the separation of chlorogenic acid and epicatechin. Integral A and ADQ diagrams and the corresponding mean diagrams complete the new concept.

HPLC determination of rhodamine B (C.I. 45170) in cosmetic products

A method is proposed for the extraction, separation, identification and quantitative measurement of rhodamine B in cosmetics samples The colour is extracted with a solution of orthophosphoric acid in either water or DMF (depending on the type of cosmetic) adsorbed on a polyamide column, and then eluted with water and methanol. Further clean-up of the extract is performed by means of a Bond Elut C-18 cartridge, from which rhodamine B is eluted with methanol-water (8∶2). The final eluate is examined by HPLC. Chromatography is performed on a C-18 column with a gradient elution from 50∶50 to 70∶30 of acetonitrile-water, containing 0.1 M sodium perchlorate, in 15 min. A diodearray detector enables peak purity analysis. The method gives recovery values of approximately 90%, or better, and the reproducibility is within 4%.

Parallel Kalman filters for peak purity analysis: extensions to non-ideal detector response

A mathematical method for detecting spectral impurities in liquid chromatographic peaks measured with a diode array detector (LC-DAD) is described. The new method compensates for certain measurement non-idealities, minimizing the likelihood of a false indication of an impurity. The algorithm, evolving projection analysis, is based on a previously reported method in which points in an n-dimensional absorbance space ( n = number of wavelengths) are projected onto two-dimensional subspaces and modeled using the Kalman filter. Model deviations can be indicative of the presence of spectral impurities, or may result from measurement artifacts such as heteroscedastic noise or non-linear detector response. The modified algorithm incorporates model variants that mask the effects of heteroscedastic noise and non-linearities so that impurities can be detected even in the presence of these artifacts. A noise model is developed for the DAD and simulations are used to evaluate the modified algorithm. Simulations are validated through the use of experimental data. It is demonstrated that, except in cases where the effects of non-linearities become severe, the modified model successfully compensates for these measurement non-idealities.

Algorithm for the assessment of peak purity in liquid chromatography with photodiode-array detection

An algorithm for peak purity analysis based on the Gram-Schmidt orthogonalization method is described. The data are pretreated by using two different normalization techniques. The performance of this approach is investigated for liquid chromatography with photodiode-array detection (LC-DAD) data. The results are compared with the ones obtained by means of fixed size window evolving factor analysis (FSW EFA). The effects of heteroscedasticity and non linearities are discussed.

Real-time principal component analysis using parallel Kalman filter networks for peak purity analysis

A new approach for pedorming principal component analysls (PCA) during data acquisition is descrlbed. The method is based on a network of multllkmar models whkh are fit to data with the dlscrete Kaiman filter. Appilcatlon to absorbance matrices such as those obtained in chromatography wlth multiwavelength detection is considered. Multivariate data projected into two- and three-dlmenslonal subspaces are fit with linear and planar models, respectively. Model deviations, detected using principles of adaptive Kalman filtering, are used to elucidate the rank of the data set. Principal component eigenvectors are then constructed from the individual models. Results of this initial work using simulated and experimental data demonstrate that extraction of the flrst two principal components is readily accomplished and eigenvectors obtained are in good agreement wlth tradltlonai batch PCA results. Extendon to more principal components should be possible although It wlli increase the number and complexity of models. Advantages of the new algorithm include its recursive implementation, parallel structure, and ability to Indicate model errors as a function of time. The procedure should prove particularly useful for self-modeling curve resolution applications In chromatography.