Insights into the selectivity of polar stationary phases based on quantitative retention mechanism assessment in hydrophilic interaction chromatography.

Hydrophilic interaction chromatography (HILIC) offers different selectivity than reversed-phase liquid chromatography (RPLC). However, our knowledge of the driving force for selectivity is limited and there is a need for a better understanding of the selectivity in HILIC. Quantitative assessment of retention mechanisms makes it possible to investigate selectivity based on understanding the underlying retention mechanisms. In this study, selected model compounds from the Ikegami selectivity tests were evaluated on different polar stationary phases. The study results revealed significant insights into the selectivity in HILIC. First, hydroxy and methylene selectivity is driven by hydrophilic partitioning; but surface adsorption for 2-deoxyuridine or 5-methyluridine reduces the selectivity factor. Furthermore, the retention of 2-deoxyuridine or 5-methyluridine by surface adsorption in combination with the phase ratio explain the difference in hydroxy or methylene selectivity observed among different stationary phases. Investigations on xanthine positional isomers (1-methylxanthine/3-methylxanthine, theophylline/theobromine) indicate that isomeric selectivity is controlled by surface adsorption; however, hydrophilic partitioning may contribute to resolution by enhancing overall retention. In addition, two pairs of nucleoside isomers (adenosine/vidarabine, 2'-deoxy and 3'-deoxyguanosine) provide an example that isomeric selectivity can also be controlled by hydrophilic partitioning if their partitioning coefficients are significantly different in HILIC. Although more data is needed, the current study provides a mechanistic based understanding of the selectivity in HILIC and potentially a new way to design selectivity tests.

A design of experiment based RP-HPLC method for the simultaneous estimation of antihypertensive drugs with greenness assessment.

The correlation between blood pressure (BP) and cardiovascular risk has a continuous, positive, and linear pattern. Lowering high BP decreases the risk associated with cardiovascular disease. Chlorthalidone (CHD) and Losartan potassium (LOS) combination is used to treat hypertension. The analytical community was concerned with minimizing or reducing the use of toxic chemicals and solvents. Therefore, the current study aimed to develop a rapid, sensitive, and cost-effective green RP-HPLC method to determine CHD and LOS simultaneously in a short analysis of time. Method optimization was performed by Central composite design (CCD), the flow rate and the change of time were chosen as factors. Effective separation was conducted on Zorbax SB-C18 (4.6 mm × 150 mm, 5 µm) column by gradient mobile phase comprising phosphate buffer and ethanol flowing at 0.859 ml/min, and the wavelength detected at 230 nm. As per ICH criteria, the technique was proven to be precise, accurate, and linear over the concentration range of 4.3-8.1 µg/ml for CHD and 35-65 µg/ml for LOS. Furthermore, the method's greenness was examined by three different metrics, confirming that less toxic effect on the environment. Hence, the optimized approach proves to be eco-friendly, simple, and robust for the concurrent evaluation of CHD and LOS in pharmaceutical formulations.

Assessment of Lipophilicity Parameters of Antimicrobial and Immunosuppressive Compounds.

Lipophilicity in addition to the solubility, acid-base character and stability is one of the most important physicochemical parameters of a compound required to assess the ADMET properties (absorption, distribution, metabolism, excretion and toxicity) of a bioactive molecule. Therefore, the subject of this work was to determine the lipophilicity parameters of selected antimicrobial and immunosuppressive compounds such as delafloxacin, linezolid, sutezolid, ceftazidime, everolimus and zotarolimus using thin-layer chromatography in reversed phase system (RP-TLC). The chromatographic parameters of lipophilicity (RMW) for tested compounds were determined on different stationary phases: RP18F254, RP18WF254 and RP2F254 using ethanol, acetonitrile, and propan-2-ol as organic modifiers of mobile phases used. Chromatographically established RMW values were compared with partition coefficients obtained by different computational methods (AlogPs, AClogP, AlogP, MlogP, XlogP2, XlogP3, logPKOWWIN, ACD/logP, milogP). Both cluster and principal component analysis (CA and PCA) of the received results allowed us to compare the lipophilic nature of the studied compounds. The sum of ranking differences analysis (SRD) of all lipophilicity parameters was helpful to select the most effective method of determining the lipophilicity of the investigated compounds. The presented results demonstrate that RP-TLC method may be a good tool in determining the lipophilic properties of studied substances. Obtained lipophilic parameters of the compounds can be valuable in the design of their new derivatives as efficient antimicrobial and immunosuppressive agents.

Rapid quantification of fatty acids in plant oils and biological samples by LC-MS.

Analysis of fatty acids (FA) in food and biological samples such as blood is indispensable in modern life sciences. We developed a rapid, sensitive and comprehensive method for the quantification of 41 saturated and unsaturated fatty acids by means of LC-MS. Optimized chromatographic separation of isobaric analytes was carried out on a C8 reversed phase analytical column (100 × 2.1 mm, 2.6 µm core-shell particle) with a total run time of 15 min with back pressure lower than 300 bar. On an old triple quadrupole instrument (3200, AB Sciex), pseudo selected reaction monitoring mode was used for quantification of the poorly fragmenting FA, yielding limits of detection of 5-100 nM. Sample preparation was carried out by removal of phospholipids and triglycerides by solid-phase extraction (non-esterified fatty acids in oils) or saponification in iso-propanol (fatty acyls). This is not only a rapid strategy for quantification of fatty acyls, but allows the direct combination with the LC-MS-based analysis of fatty acid oxidation products (eicosanoids and other oxylipins) from the same sample. The concentrations of fatty acyls determined by means of LC-MS were consistent with those from GC-FID analysis demonstrating the accuracy of the developed method. Moreover, the method shows high precisions with a low intra-day (≤ 10% for almost all fatty acids in plasma and ≤ 15% in oils) and inter-day as well as inter-operator variability (< 20%). The method was successfully applied on human plasma and edible oils. The possibility to quantify non-esterified fatty acids in samples containing an excess of triacylglycerols and phospholipids is a major strength of the described approach allowing to gain new insights in the composition of biological samples.

QbD-steered development and validation of an RP-HPLC method for quantification of ferulic acid: Rational application of chemometric tools.

The present work describes the systematic development of a simple, rapid, sensitive, robust, effective and cost-effective reversed-phase high performance liquid chromatographic method for quantitative analysis of ferulic acid using analytical quality by design paradigms. Initially, apt wavelength for the analysis of ferulic acid was selected employing principal component analysis as the chemometric tool. An Ishikawa fishbone diagram was constructed to delineate various plausible variables influencing analytical target profile, viz. peak area, theoretical plate count, retention time and peak tailing as the critical analytical attributes. Risk assessment using risk estimation matrix and factor screening studies employing Taguchi design aided in demarcating two critical method parameters, viz. mobile phase ratio and flow rate affecting critical analytical attributes. Subsequently, the optimum operational conditions of the liquid chromatographic method were delineated using face-centred composite design. Multicollinearity among the chosen factors for optimization was analyzed by the magnitude of variance inflation factor optimized analytical design space, providing optimum method performance, was earmarked using numerical and graphical optimization and corroborated using Monte Carlo simulations. Validation, as per the ICH Q2(R1) guidelines, ratified the efficiency and sensitivity of the developed novel analytical method of ferulic acid in the mobile phase and the human plasma matrix. The optimal method used a mobile phase, comprising of acetonitrile: water (47:53% v/v, pH adjusted to 3.0 with glacial acetic acid), at a flow rate of 0.8 mL·min-1, at a λmax of 322 nm using a C18 column. Use of principal component analysis unearthed the suitable wavelength for analysis, while analytical quality by design approach, along with Monte Carlo simulations, facilitated the identification of influential variables in obtaining the "best plausible" validated chromatographic solution for efficient quantification of ferulic acid.

An application of QSRR approach and multiple linear regression method for lipophilicity assessment of flavonoids.

The analysis of quantitative structure-retention relationships (QSRR) is useful tool for assessment of compound's lipophilicity/hydrophobicity due to similarity between its retention in chromatographic system and ability to permeation through biological membranes. The main goal of this study was to compare usefulness of two reversed-phase chromatographic columns (Synergy POLAR and Synergy-FUSION) for lipophilicity assessment of 30 structurally diverse flavonoids using the QSRR approach and multiple linear regression method. The developed MLR models included the mechanistically interpretable geometrical descriptors: 3D Molecule Representation of Structure based on Electron diffraction (3D-MoRSE) and Radial Distribution Function (RDF). Both models were evaluated by the internal and external validation and selected descriptors were further interpreted. According to obtained results the FUSION-RP column can be recommended to log kw prediction of flavonoids. The comprehensive interpretation of molecular descriptors was used to present the molecular mechanisms and structural features governing the chromatographic retention of tested compounds.

Development of Two Analytical Methods Based on Reverse Phase Chromatographic and SDS-PAGE Gel for Assessment of Deglycosylation Yield in N-Glycan Mapping.

N-lined glycosylation is one of the critical quality attributes (CQA) for biotherapeutics impacting the safety and activity of drug product. Changes in pattern and level of glycosylation can significantly alter the intrinsic properties of the product and, therefore, have to be monitored throughout its lifecycle. Therefore fast, precise, and unbiased N-glycan mapping assay is desired. To ensure these qualities, using analytical methods that evaluate completeness of deglycosylation is necessary. For quantification of deglycosylation yield, methods such as reduced liquid chromatography-mass spectrometry (LC-MS) and reduced capillary gel electrophoresis (CGE) have been commonly used. Here we present development of two additional methods to evaluate deglycosylation yield: one based on LC using reverse phase (RP) column and one based on reduced sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE gel) with offline software (GelAnalyzer). With the advent of rapid deglycosylation workflows in the market for N-glycan profiling replacing overnight incubation, we have aimed to quantify the level of deglycosylation in a selected rapid deglycosylation workflow. Our results have shown well resolved peaks of glycosylated and deglycosylated protein species with RP-LC method allowing simple quantification of deglycosylation yield of protein with high confidence. Additionally a good correlation, ≥0.94, was found between deglycosylation yields estimated by RP-LC method and that of reduced SDS-PAGE gel method with offline software. Evaluation of rapid deglycosylation protocol from GlycanAssure™ HyPerformance assay kit performed on fetuin and RNase B has shown complete deglycosylation within the recommended protocol time when evaluated with these techniques. Using this kit, N-glycans from NIST mAb were prepared in 1.4 hr and analyzed by hydrophilic interaction chromatography (HILIC) ultrahigh performance LC (UHPLC) equipped with a fluorescence detector (FLD). 37 peaks were resolved with good resolution. Excellent sample preparation repeatability was found with relative standard deviation (RSD) of <5% for peaks with >0.5% relative area.

Development of a stability- indicating HPLC method for simultaneous determination of ten related substances in vonoprazan fumarate drug substance.

Vonoprazan fumarate is a novel potassium-competitive acid blocker for the treatment of acid-related diseases. In the present study, a simple, fast, and economic reversed-phase liquid chromatography (LC) method was developed for the analysis of ten related substances (raw materials, by-products and degradants) in vonoprazan fumarate. The optimized separation was performed on a Phenomenex Kinetex EVO C18 (250mm×4.6mm, 5.0µm) column. The mobile phase consisted of (A) 0.03M sodium phosphate buffer (pH adjusted to 6.5) - methanol - acetonitrile (72:25:3, v/v/v) and (B) 0.03M sodium phosphate buffer (pH adjusted to 6.5) - acetonitrile (30:70, v/v). Detection of the analytes was conducted at 230nm using a UV detector. The stability-indicating ability of this method was demonstrated by carrying out forced degradation studies. Vonoprazan underwent significant degradation when subjected to alkaline and oxidative stress conditions, while the drug proved to be stable to acidic, thermal and photolytic degradation. The degradants did not interfere with the detection of vonoprazan fumarate and its impurities. The performance of this method was validated in accordance to the regulatory guidelines recommended by the International Conference on Harmonisation (ICH) and this validation included specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision and robustness. The method proposed in this paper could be applied for process development as well as quality assurance of vonoprazan in bulk drug, since no monograph is available in official compendia.

The comparative study of micellar TLC and RP-TLC as potential tools for lipophilicity assessment based on QSRR approach.

Lipophilicity of compound is well known as vital physicochemical property of a molecule, which determines its biological activity. Nonetheless, the assessment of a lipophilicity is still problematic and focuses attention of scientists. Although, the shake-flask method is still considered as a gold standard for experimental determination of lipophilicity, currently the chromatographic approach is mostly used. Among chromatographic methods used for lipophilicity assessment, thin layer chromatography (TLC) is still one of the most popular tools. The main goal of this study was to compare classical reversed-phase thin layer chromatography (RP-TLC) and micellar TLC as potential tools for lipophilicity assessment. Micellar liquid chromatography has significantly lover environment impact than classical reversed-phase liquid chromatography. Additionally comparison of cationic and anionic surfactants (CTAB and SDS), which have different chemical properties, as modifiers of mobile phase in micellar TLC were investigated. The Quantitative Structure-Retention Relationships (QSRR) approach was used in order to present molecular mechanisms of retention in investigated chromatographic systems. The study was based on chemically diverse model set compounds, with a proved therapeutic or toxic potential. According to obtained results the micellar TLC with CTAB as surfactant can be recommended to logP prediction. The obtained QSRR models indicated that adsorption of CTAB monomers on CN modified surface of silica gel and the silanol-quaternary ammonium interactions are possible. Consequently, the reduction of interaction between molecules and free silanol, contributes to the improvement of logP predictions. These result were confirmed by regression and classification methods.

Optimization and validation of reverse phase HPLC method for qualitative and quantitative assessment of polyphenols in seaweed.

A simple reverse phase-high performance liquid chromatography (RP-HPLC) coupled to a diode array detector (DAD) and negative ion electrospray mass spectrometer (ESI-MS) method was developed for simultaneous identification and quantification of phenolic antioxidants in seaweed. The proposed method was validated in terms of linearity, limits of detection (LOD), limits of quantification (LOQ), recovery and intermediate precision. The calibration curves were linear with correlation coefficient ranging from 0.9909 to 0.9997 while the values of LOD (0.26-0.82mg/L), LOQ (0.77-2.50mg/L), recovery (≥97.2%) and precision in terms of retention time (%RSD ≤2.27) and peak area (% RSD ≤5.11) were satisfactory. Brown seaweed Himanthalia elongata used in this study was extracted with 60% methanol and the crude extract was cleaned with SPE (Solid Phase Extraction) cartridge. HPLC-DAD-MS/MS analysis of the SPE fraction allowed the identification of 7 phenolic compounds comprising phlorotannins, hydroxybenzoic acid, hydroxycinnamic acid and flavonols subclasses of polyphenols. Quantitative analysis of these compounds revealed the presence of phloroglucinol (394.1±4.33µg/g), gallic acid (96.3±3.12µg/g), chlorogenic acid (38.8±1.94µg/g), caffeic acid (44.4±2.72µg/g), ferulic acid (17.6±0.85µg/g), myricetin (8.6±0.85µg/g) and quercetin (4.2±0.15µg/g), in the extract. The SPE fraction were tested for antioxidant capacity which were significantly (P <0.05) higher (EC50; 14.5±0.57mg/g) than the ascorbic acid (EC50; 35.8±0.59mg/g) and the crude extract (EC50; 46.3±0.48mg/g). The occurrence of all these phenolic antioxidant compounds in H. elongata extract suggested that the developed method is sensitive enough and reproducible and could be used for qualitative and quantitative assessment of polyphenols in seaweed.

Assessment of Lipophilicity Indices Derived from Retention Behavior of Antioxidant Compounds in RP-HPLC.

Reverse phase high pressure liquid chromatography was employed in order to evaluate the lipophilicity of antioxidant compounds from different classes, such as phenolic acids, flavanones, flavanols, flavones, anthocyanins, stilbenes, xantonoids, and proanthocyanidins. The retention time of each compound was measured using five different HPLC columns: RP18 (LiChroCART, Purosphere RP-18e), C8 (Zorbax, Eclipse XDBC8), C16-Amide (Discovery RP-Amide C16), CN100 (Saulentechnik, Lichrosphere), and pentafluorophenyl (Phenomenex, Kinetex PFP), and the mobile phase consisted of methanol and water (0.1% formic acid) in different proportions. The measurements were conducted at two different column temperatures, room temperature (22 °C) and, in order to mimic the environment from the human body, 37 °C. Furthermore, principal component analysis (PCA) was used to obtain new lipophilicity indices and holistic lipophilicity charts. Additionally, highly representative depictions of the chromatographic behavior of the investigated compounds and stationary phases at different temperatures were obtained using two new chemometric approaches, namely two-way joining cluster analysis and sum of ranking differences.

Assessment of the chromatographic lipophilicity of eight cephalosporins on different stationary phases.

The retention behaviors were investigated for a series of eight cephalosporins in thin-layer chromatography (TLC) using stationary phases of RP-2, RP-8, RP-18, NH2, DIOL, and CN chemically bonded silica gel. Additionally, various binary mobile phases (water/methanol and water/acetone) were used in different volume proportions. The retention behavior of the analyzed molecules was defined by RM0 constant. In addition, reversed phase high performance liquid chromatography (RP-HPLC) was performed in lipophilicity studies by using immobilized artificial membrane (IAM) stationary phase. Obtained chromatographic data (RM0 and logk'IAM) were correlated with the lipophilicity, expressed as values of the log calculated (logPcalc) and experimental (logPexp(shake-flask)) partition coefficient. Principal component analysis (PCA) was applied in order to obtain an overview of similarity or dissimilarity among the analyzed compounds. Hierarchical cluster analysis (HCA) was performed to compare the separation characteristics of the applied stationary phases. This study was undertaken to identify the best chromatographic system and chromatographic data processing method to enable the prediction of logP values. A comprehensive chromatographic investigation into the retention of the analyzed cephalosporins revealed a similar behavior on RP-18, RP-8 and CN stationary phases. The weak correlations obtained between experimental and certain computed lipophilicity indices revealed that RM0 and PC1/RM are relevant lipophilicity parameters and the RP-8, CN and RP-18 plates are appropriate stationary phases for lipophilicity investigation, whereas computational approaches still cannot fully replace experimentation.

Economical, Plain, and Rapid Authentication of Actaea racemosa L. (syn. Cimicifuga racemosa, Black Cohosh) Herbal Raw Material by Resilient RP-PDA-HPLC and Chemometric Analysis.

INTRODUCTION: The medicinal plant Actaea racemosa L. (Ranunculaceae, aka black cohosh) is widely used to treat climacteric complaints as an alternative to hormone substitution. Recent trials prove efficacy and safety of the approved herbal medicinal products from extracts of pharmaceutical quality. This led to worldwide increasing sales. A higher demand for the plant material results in problems with economically motivated adulteration. Thus, reliable tools for herbal drug authentication are necessary. OBJECTIVE: To develop an economical, plain, and rapid method to distinguish between closely related American and Asian Actaea species, using securely established and resilient analytical methods coupled to a chemometric evaluation of the resulting data. METHODOLOGY: We developed and validated a RP-PDA-HPLC method including an extraction by ultra-sonication to determine the genuine contents of partly hydrolysis-sensitive polyphenols in Actaea racemosa roots and rhizomes, and applied it to a large number of 203 Actaea samples consisting of seven species. RESULTS: We were able to generate reliable data with regards to the polyphenolic esters in the samples. The evaluation of this data by principle component analysis (PCA) made a discrimination between Asian Actaea species (sheng ma), one American Actaea species (Appalachian bugbane), and A. racemosa possible. CONCLUSION: The developed RP-PDA-HPLC method coupled to PCA is an excellent tool for authentication of the Actaea racemosa herbal drug, and can be a powerful addition to the TLC methods used in the dedicated pharmacopoeias, and is a promising alternative to expensive and lots of expertise requiring methods. Copyright © 2016 John Wiley & Sons, Ltd.

Multivariate assessment of lipophilicity scales-computational and reversed phase thin-layer chromatographic indices.

Needs for fast, yet reliable means of assessing the lipophilicities of diverse compounds resulted in the development of various in silico and chromatographic approaches that are faster, cheaper, and greener compared to the traditional shake-flask method. However, at present no accepted "standard" approach exists for their comparison and selection of the most appropriate one(s). This is of utmost importance when it comes to the development of new lipophilicity indices, or the assessment of the lipophilicity of newly synthesized compounds. In this study, 50 well-known, diverse compounds of significant pharmaceutical and environmental importance have been selected and examined. Octanol-water partition coefficients have been measured with the shake-flask method for most of them. Their retentions have been studied in typical reversed thin-layer chromatographic systems, involving the most frequently employed stationary phases (octadecyl- and cyano-modified silica), and acetonitrile and methanol as mobile phase constituents. Twelve computationally estimated logP-s and twenty chromatographic indices together with the shake-flask octanol-water partition coefficient have been investigated with classical chemometric approaches-such as principal component analysis (PCA), hierarchical cluster analysis (HCA), Pearson's and Spearman's correlation matrices, as well as novel non-parametric methods: sum of ranking differences (SRD) and generalized pairwise correlation method (GPCM). Novel SRD and GPCM methods have been introduced based on the Comparisons with One VAriable (lipophilicity metric) at a Time (COVAT). For the visualization of COVAT results, a heatmap format was introduced. Analysis of variance (ANOVA) was applied to reveal the dominant factors between computational logPs and various chromatographic measures. In consensus-based comparisons, the shake-flask method performed the best, closely followed by computational estimates, while the chromatographic estimates often overlap with in silico assessments, mostly with methods involving octadecyl-modified silica stationary phases. The ones that employ cyano-modified silica perform generally worse. The introduction of alternative coloring schemes for the covariance matrices and SRD/GPCM heatmaps enables the discovery of intrinsic relationships among lipophilicity scales and the selection of best/worst measures. Closest to the recommended logKOW values are ClogP and the first principal component scores obtained on octadecyl-silica stationary phase in combination with methanol-water mobile phase, while the usage of slopes derived from Soczewinski-Matyisik equation should be avoided.

Development of a rapid RP-UHPLC-MS method for analysis of modifications in therapeutic monoclonal antibodies.

Chemical or enzymatic modifications of therapeutic monoclonal antibodies (MAbs) that have high risk to safety and efficacy are defined as critical quality attributes (CQAs). During therapeutic MAbs process development, thorough characterization and quantitative monitoring of CQAs requires a variety of analytical techniques. This paper describes the development of a rapid analytical method to assess modifications in MAbs, based on the analysis of subdomains with molecular weights of ∼25kDa. These subdomains were generated by digestion with a highly specific IdeS protease, followed by disulfide bond reduction. A reversed phase UHPLC-MS method was developed that provides efficient separation and identification of the subdomains (Fc, LC, and Fd) and related variants within 10min. Sample preparation and UHPLC instrument parameters were systematically evaluated. The methodology was applied to MAb stress panel characterization to capture the degradations induced by various stress conditions. Among the CQAs monitored by this method, Fc oxidation levels were compared with the values obtained by the more complicated and time-consuming peptide mapping method. The similar trends observed by the two methods demonstrated that the IdeS-UHPLC method is valuable as a higher throughput alternative to peptide mapping for monitoring modifications. In particular, a high-throughput methodology is preferred for analysis of the many samples associated with process development studies. Overall the method has been demonstrated as a fast, convenient and informative platform approach for analysis of therapeutic MAbs modifications including CQAs.

Structural Analysis and Quantitative Determination of Clevidipine Butyrate Impurities Using an Advanced RP-HPLC Method.

Eleven potential impurities, including process-related compounds and degradation products, have been analyzed by comprehensive studies on the manufacturing process of clevidipine butyrate. Possible formation mechanisms could also be devised. MS and NMR techniques have been used for the structural characterization of three previously unreported impurities (Imp-3, Imp-5 and Imp-11). To separate and quantify the potential impurities in a simultaneous fashion, an efficient and advanced RP-HPLC method has been developed. In doing so, four major degradation products (Imp-2, Imp-4, Imp-8 and Imp-10) can be observed under varying stress conditions. This analytical method has been validated according to ICH guidelines with respect to specificity, accuracy, linearity, robustness and stability. The method described has been demonstrated to be applicable in routine quality control processes and stability evaluation studies of clevidipine butyrate.

Gradient liquid chromatographic retention time prediction for suspect screening applications: A critical assessment of a generalised artificial neural network-based approach across 10 multi-residue reversed-phase analytical methods.

For the first time, the performance of a generalised artificial neural network (ANN) approach for the prediction of 2492 chromatographic retention times (tR) is presented for a total of 1117 chemically diverse compounds present in a range of complex matrices and across 10 gradient reversed-phase liquid chromatography-(high resolution) mass spectrometry methods. Probabilistic, generalised regression, radial basis function as well as 2- and 3-layer multilayer perceptron-type neural networks were investigated to determine the most robust and accurate model for this purpose. Multi-layer perceptrons most frequently yielded the best correlations in 8 out of 10 methods. Averaged correlations of predicted versus measured tR across all methods were R(2)=0.918, 0.924 and 0.898 for the training, verification and test sets respectively. Predictions of blind test compounds (n=8-84 cases) resulted in an average absolute accuracy of 1.02±0.54min for all methods. Within this variation, absolute accuracy was observed to marginally improve for shorter runtimes, but was found to be relatively consistent with respect to analyte retention ranges (~5%). Finally, optimised and replicated network dependency on molecular descriptor data is presented and critically discussed across all methods. Overall, ANNs were considered especially suitable for suspects screening applications and could potentially be utilised in bracketed-type analyses in combination with high resolution mass spectrometry.

DETERMINATION OF TENOXICAM IN THE PLASMA BY REVERSE PHASE HPLC METHOD USING SINGLE STEP EXTRACTION TECHNIQUE: A RELIABLE AND COST EFFECTIVE APPROACH.

A simple and cost effective RPLC-UV bio-analytical method was developed and used for tenoxicam quantification on ODS Hypersil C-18 column using classical liquid-liquid extraction technique for sample preparation. Acetonitrile was used as precipitating agent for plasma proteins and supernatant was taken for injection without any further modification. The bio-analytical method depends upon isocratic elution using binary mixture of aqueous 0.1 M potassium dihydrogen phosphate and acetonitrile in 6 : 4 ratio. The pH of mobile phase was adjusted to 2.8 which favor tenoxicam to remain undissociated throughout the analysis. The optimized flow rate of 1.0 mL/min provided proper separation of peaks and column clean up within 5 min. The UV detection was achieved at 381 nm and 4.29 min. Reproducible calibration curve gave 0.325 µg/mL LOQ, linear dynamic range from 0.325 to 20 µg/mL and recovery from plasma was 98.5% with %CV 0.2314 achieved. After validation, the method was applied in pharmacokinetic study in healthy human volunteers (n = 8). The pharmacokinetic parameters were evaluated using kinetica version 4.1.1. The values of C. and area under curve for current study were 1.776 ± 0.003 pg/mL and 179.97 ± 0.0681 (mean ± SEM) pg x h/mL. The values of t, and volume of distribution for tenoxicam in current study were 74.103 0.167 h (mean ± SEM) and 11.962 ± 0.0677 L/kg (mean ± SEM), respectively. This method was simple, sensitive and successfully applied in pharmacokinetic studies. It can be extended to bioequivalence studies and evaluation of tenoxicam in different clinical situations.

LIPOPHILICITY ASSESSMENT OF SPIRONOLACTONE BY MEANS OF REVERSED PHASE LIQUID CHROMATOGRAPHY AND BY NEWLY DEVELOPED CALCULATION PROCEDURES.

The parameters of lipophilicity of spironolactone (a single member of steroids group), which is widely applied as diuretic and antihypertensive agent, were experimentally determined by reversed-phase TLC and HPLC methods as well as calculated using different computer programs and also by a novel mode based on topological indices. Various stationary phases, such as RP-18WFA4254, RP-2F254, RP-18F254 and also different binary solvent systems composed of organic modifier (e.g., methanol, dioxane, acetone) and water were used as mobile phases in order to predict the following chromatographic parameters: Rm, and logkw, respectively. LogP of examined spironolactone calculated with respective theoretical procedures: AlogPs, logPKOWWIN, xlogP2, xlogP3, AClogP, AlogP, MlogP and also logPaverage were obtained from online package software. The partition coefficients expressed as logP1, logP2 and logP3 were calculated by means of the formulae based on the numerical values of the following topological indices: , 0B, 1B, W, 0χv and IB, which was novelty of this study. A good agreement between logP calculated by new method and experimentally estimated lipophilicity parameters (by chromatography and by shake flask method) was found. The results confirmed applicability of the topological indices for calculating lipophilicity of spironolactone as alternative procedure to the experimental and other computed logP values.

Performance comparison of partial least squares-related variable selection methods for quantitative structure retention relationships modelling of retention times in reversed-phase liquid chromatography.

The relative performance of six multivariate data analysis methods derived from or combined with partial least squares (PLS) has been compared in the context of quantitative structure-retention relationships (QSRR). These methods include, GA (genetic algorithm)-PLS, Monte Carlo uninformative variable elimination (MC-UVE), competitive adaptive reweighted sampling (CARS), iteratively retaining informative variables (IRIV), variable iterative space shrinkage approach (VISSA) and PLS with automated backward selection of predictors (autoPLS). A set of 825 molecular descriptors was computed for 86 suspected sports doping compounds and used for predicting their gradient retention times in reversed-phase liquid chromatography (RPLC). The correlation between molecular descriptors selected by each technique and the retention time was established using the PLS method. All models derived from a selected subset of descriptors outperformed the reference PLS model derived from all descriptors, with very small demands of computational time and effort. A performance comparison indicated great diversity of these methods in selecting the most relevant molecular descriptors, ranging from 28 for CARS to 263 for MC-UVE. While VISSA provided the lowest degree of over-fitting for the training set, CARS demonstrated the best compromise between the prediction accuracy and the number of selected descriptors, with the prediction error of as low as 46s for the external test set. Only ten descriptors were found to be common for all models, with the characteristics of these descriptors being representative of the retention mechanism in RPLC.