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.

Selective Stability Indicating Liquid Chromatographic Method Based on Quality by Design Framework and In Silico Toxicity Assessment for Infigratinib and Its Degradation Products.

Infigratinib, a protein kinase inhibitor employed in the therapeutic management of cholangiocarcinoma, was subjected to various stress conditions, including hydrolytic (acidic and alkaline), oxidative, photolytic, and thermal stress, in accordance with the rules established by the International Council for Harmonization. A cumulative count of five degradation products was observed. The application of the Quality by Design principle was utilized in the development of a rapid and specific separation method for Infigratinib and its degradation products. The methodology employed in this study was derived from an experimental design approach, which was utilized to examine the critical process parameters associated with chromatographic systems. The reversed-phase high-performance liquid chromatography technique, employing a C18 column and a mobile phase composed of a gradient mixture of 25 mM ammonium acetate buffer at pH 6.0 and acetonitrile, successfully facilitated the chromatographic separation. The methodology was expanded to include the utilization of UPLC-quadrupole tandem mass spectrometry in order to conduct a comprehensive analysis of the structural properties and characterize the degradation products. Overall, five degradation products were found in different stress conditions. The method was verified at certain working points, wherein a linearity range (5.0-200.0 µg/mL) was developed and other parameters such as accuracy, repeatability, selectivity, and system suitability were evaluated. Finally, the toxicity and mutagenicity of Infigratinib and its degradation products were predicted using in silico software, namely DEREK Nexus® (version 6.2.1) and SARAH Nexus® (version 3.2.1). Various toxicity endpoints, including chromosomal damage, were predicted. Additionally, two degradation products were also predicted to be mutagenic.

The Effect of Milling on the Ethanolic Extract Composition of Dried Walnut (Juglans regia L.) Shells.

This study investigates the ethanolic extract of dried walnut (Juglans regia L.) shells upon hammer milling (HM) and ball milling (BM) grinding processes. Marked differences were observed in the attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectra. The two extracts were investigated by reversed-phase liquid chromatography coupled with electrospray ionization and high-resolution mass spectrometry (RPLC-ESI-HRMS). Following enzymatic digestion, the fatty acids (FAs) were examined, and tandem MS of epoxidized species was applied to establish the C-C double bond position; the most abundant species were FA 18:2 Δ9,12, FA 18:1 Δ9, and FA 18:3 Δ9,12,15. However, no significant qualitative differences were observed between FAs in the two samples. Thus, the presence of potential active secondary metabolites was explored, and more than 30 phenolic compounds, including phenols, ellagic acid derivatives, and flavonoids, were found. Interestingly, the HM samples showed a high concentration of ellagitannins and hydrolyzable tannins, which were absent in the BM sample. These findings corroborate the greater phenolic content in the HM sample, as evaluated by the Folin-Ciocalteu test. Among the others, the occurrence of lanceoloside A at m/z 391.1037 [C19H20O9-H]-, and a closely related benzoyl derivate at m/z 405.1190 (C20H22O9-H]-), was ascertained. The study provides valuable information that highlights the significance of physical pre-treatments, such as mill grinding, in shaping the composition of extracts, with potential applications in the biorefinery or pharmaceutical industries.

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.

Investigation into reversed-phase chromatography peptide separation systems part V: Establishment of a screening strategy for development of methods for assessment of pharmaceutical peptides' purity.

The paper describes a simple and rapid reversed-phase UHPLC method development screening strategy for the purity determination of peptide-based pharmaceuticals. The protocol utilises five disparate column and six volatile or non-volatile mobile phases (i.e., 30 combinations). The method development strategy has been demonstrated to be highly effective in identifying conditions which generate complementary selectivity and good peak shape. Columns with varying degrees of charge (positive and negative), in addition to their differing hydrophobic character, were used in combination with mobile phases within the pH range of 2.3 to 5.1. The novel ion-pair / chaotropic reagent ammonium hexafluorophosphate at pH 2.3 was shown to be an extremely useful mobile phase additive in that it produced excellent complementary separation and good peak shape. Methanesulfonic acid was demonstrated to be a good alternative to the ubiquitously employed trifluoroacetic acid which failed to generate optimum separation for the peptides investigated highlighting the importance of screening disparate mobile phase additives. Both ammonium hexafluorophosphate and methanesulfonic acid were shown not to adversely affect the stability of C18 columns or demonstrated any irreversible adsorption / memory effects. No pH hysteresis effects were demonstrated with any of the stationary phases on mobile phase pH cycling. No major problems have been observed with the novel mobile phase additives ammonium hexafluorophosphate and methanesulfonic acid, however, it is recommended that they be used with caution until long-term routine use has been established.

Green NP-HPTLC and green RP-HPTLC methods for the determination of thymoquinone: A contrast of validation parameters and greenness assessment.

INTRODUCTION: Thymoquinone (TQ) is a naturally derived bioactive compound with several therapeutic effects. OBJECTIVE: The highly sensitive, rapid and green normal-phase (NP)/reversed-phase (RP) high-performance thin-layer chromatography (HPTLC) densitometry technique was developed for the determination of TQ in various plant extracts of different geographical regions, commercial capsules, creams and essential oils. METHODOLOGY: The NP densitometry estimation of TQ was performed using a cyclohexane-ethyl acetate (90:10, v/v) green solvent system, while, the RP-densitometry estimation of TQ was performed using an ethanol-water (80:20, v/v) green solvent system. The estimation of TQ was conducted at 259 nm. RESULTS: The NP and RP densitometry techniques were observed linear in the range of 25-1000 and 50-600 ng/band, respectively. All validation parameters such as accuracy, precision, robustness and sensitivity of NP/RP densitometry were observed within the limit of regulatory requirements and hence found to be suitable for the determination of TQ. The TQ contents were found to be highest in the Saudi Arabian extract followed by the Syrian extract, Indian extract, commercial capsules, commercial creams, Jordanian extract, Egyptian extract, Palestinian extract and commercial essential oils using NP densitometry. The TQ contents were found in same order using RP densitometry, but they were much lower than those recorded using NP densitometry. The Analytical GREEnness (AGREE) scores of NP and RP densitometry were found to be 0.82 and 0.84, respectively, suggesting an excellent greenness profile. CONCLUSIONS: Based on these results, NP/RP densitometry was found to be suitable for the pharmaceutical assay of TQ.

Multivariate assessment of anticancer oleanane triterpenoids lipophilicity.

Naturally occurring molecules are excellent sources of lead compounds. A series of oleanolic acid (OA) derivatives previously synthesized in our laboratory, which show promising antitumor activity, have been analyzed in terms of lipophilicity evaluation applying chromatographic and computational approaches. Retention data obtained on three reversed-phase liquid chromatography stationary phases (RP-HPLC) and immobilized artificial membrane chromatography (IAM-HPLC) were compared with computational methods using chemometric tools such as cluster analysis, principal component analysis and sum of ranking differences. To investigate the molecular mechanism of retention quantitive structure retention relationship analysis was performed, based on the genetic algorithm coupled with multiple linear regression (GA-MLR). The obtained results suggested that the ionization potential of studied molecules significantly affects their retention in classical RP-HPLC. In IAM-HPLC additionally, polarizability-related descriptors also play an essential role in that process. The lipophilicity indices comparison shows significant differences between the computational lipophilicity and chromatographically determined ones.

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.

A comprehensive stability assessment of insulin degludec using New customized validated RP-HPLC and SEC-HPLC methods in an orthogonal testing protocol.

Stress testing of biopharmaceuticals plays an important role in preparation of their stability profiles through investigation of possible degradation pathways and identification of degradation products, so in this study Insulin Degludec which is a new generation ultra-long-acting basal insulin is subjected to stress conditions as different temperatures, different pH, oxidation, mechanical agitation, and repeated freeze and thaw cycles to generate possible degradation products and aggregation that are investigated by two new validated RP-HPLC and SEC-HPLC methods in addition to dynamic light scattering (DLS) and native polyacrylamide gel electrophoresis (Nu-PAGE). SEC-HPLC was used to investigate formation of aggregates whose results were correlated with those obtained from DLS and Nu-PAGE, while RP-HPLC was used to investigate any possible chemical modifications. The Proposed RP-method had limit of detection (LOD) and limit of quantitation (LOQ) of 0.012 mg/mL and 0.045 mg/mL respectively and accuracy of 99.22 ± 1.07 %, while the SEC methods had limit of detection (LOD) and limit of quantitation (LOQ) of 0.012 mg/mL and 0.031 mg/mL, respectively. The degradation pattern due to high temperature effect and oxidation is investigated by LC- tandem mass spectrometry. Results showed that Insulin degludec is highly stable under low temperature, mechanical agitation and repeated freeze and thaw stress conditions but elevated temperature and high acidic condition lead to formation of aggregates and also chemical modifications including deamidation, isomerization and oxidation. Such different chemical degradation pathways are due to presence of variable reactive moieties in Insulin degludec structure. Insulin degludec is highly vulnerable to oxidation at the sulfur containing cysteine residue in B chain in position B7 forming trioxidation derivative when exposed to hydrogen peroxide. Formation of A21-Asp and A18-Asp deamidated variants as well as B3-Asp and B3-isoAsp deamidated variants are prominent degradation pathways at neutral pH but at elevated temperature.

Preparation and characterization of 96-well microplates coated with molecularly imprinted polymer for determination and biosimilarity assessment of recombinant human erythropoietin.

Synthesis and applications of molecularly imprinted polymers (MIP) are rapidly growing. In this study, a biomimetic MIP was prepared through silanes polymerization on the surface of 96-well microplates using recombinant human erythropoietin-alfa (rhEPO) as a template molecule. The rhEPO was immobilized onto the plate surface using bi-functional cross-linker and a thin imprinted layer following sol-gel procedure was constructed. After template extraction, uniform three-dimensional cavities compatible with the configuration of rhEPO were obtained. The rhEPO-MIP preparation was optimized using 2-level factorial design and response surface design where polymerization time and interactions between the different variable were found to be the most significant factors. Size-exclusion chromatography (SEC) was used to monitor the stability of the rhEPO under the investigated polymerization conditions. Determination of rhEPO using the MIP microplate showed good dynamic response fitting to the 4 PL regression model (0.9962) over a concentration range of 10.00 - 100.00 ng mL-1. Adsorption of rhEPO onto MIP followed the Langmuir isotherm model (r = 0.9957, χ2 =0.02786) with pseudo-second-order kinetics (r = 0.9984). The surface of the rhEPO-MIP was characterized using scanning electron microscopy (SEM) while step-by-step surface modification was tracked using Fourier transform infrared (FTIR) spectroscopy. The rhEPO-MIP was able to distinguish between the rhEPO-alfa template and modified rhEPO molecules; rhEPO-beta, hyperglycosylated and pegylated forms (imprinting factors < 2) and in the commonly used formulation additive human serum albumin (HSA) (R% = 113.96 -95.22%). The rhEPO-MIP was applied to compare the receptor-binding pattern to rhEPO and its biosimilars / structural analogues. The results were cross-validated using the conventional assay protocol (RP-HPLC and ELISA) and an acceptable correlation was observed with RP-HPLC (maximum deviation is 7.78%). This work confirmed the applicability of rhEPO-MIP with its unique binding features for batch release, stability and biosimilarity assessment as well as subsequent evaluation of batch-to-batch consistency during bioproduction of target analytes.

Assessment and application of Marfey's reagent and analogs in enantioseparation: a decade's perspective.

Of the various methods available for high-performance liquid chromatography separation of enantiomers (of e.g. amino acids and amino group containing compounds) by the pre-column derivatization approach, use of Marfey's reagent has been most successful with continued application since its introduction in 1984. The reagent is prepared from difluoro dinitro benzene by nucleophilic substitution of one of its F atoms by l-alanine amide. There is flexibility to prepare several chiral variants (by substituting the F atom with different chiral auxiliaries) and to tailor the hydrophobicity and resolution, ultimately, of the diastereomeric derivatives. The present paper assesses and reviews applications of Marfey's reagent and its chiral variants (i.e. other FDNP reagents) for enantioseparation of certain amino group containing drugs/amino acids, and to provide some case studies on enantiomeric separations that are important for the pharmaceutical industry. Various explanations for separation mechanism and elution order using FDNP reagents are included and the question of the configuration of the corresponding enantiomer using an indirect approach has also been addressed.

Rapid Screening α-Glucosidase Inhibitors from Natural Products by At-Line Nanofractionation with Parallel Mass Spectrometry and Bioactivity Assessment.

In this study, a novel at-line nanofractionation screening platform was successfully developed for the rapid screening and identification of α-glucosidase inhibitors from natural products. A time-course bioassay based on high density well-plates was performed in parallel with high resolution mass spectrometry (MS), providing a straightforward and rapid procedure to simultaneously obtain chemical and biological information of active compounds. Through multiple nanofractionations into the same well-plate and comparisons of the orthogonal separation results of hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography (RPLC), the α-glucosidase inhibitors can be accurately identified from co-eluates. The screening platform was comprehensively evaluated and validated, and was applied to the screenings of green tea polyphenols and Ginkgo folium flavonoids. After accurate peak shape and retention time matching between the bioactivity chromatograms and MS chromatograms, ten α-glucosidase inhibitors were successfully screened out and identified. The proposed screening method is rapid, effective and can avoid ignoring low abundant/active inhibitors.

A Standardized Strategy for Simultaneous Quantification of Urine Metabolites to Validate Development of a Biomarker Panel Allowing Comprehensive Assessment of Dietary Exposure.

SCOPE: Metabolites derived from individual foods found in human biofluids after consumption could provide objective measures of dietary intake. For comprehensive dietary assessment, quantification methods would need to manage the structurally diverse mixture of target metabolites present at wide concentration ranges. METHODS AND RESULTS: A strategy for selection of candidate dietary exposure biomarkers is developed. An analytical method for 62 food biomarkers is validated by extensive analysis of chromatographic and ionization behavior characteristics using triple quadrupole mass spectrometry. Urine samples from two food intervention studies are used: a controlled, inpatient study (n = 19) and a free-living study where individuals (n = 15) are provided with food as a series of menu plans. As proof-of-principle, it is demonstrated that the biomarker panel could discriminate between menu plans by detecting distinctive changes in the concentration in urine of targeted metabolites. Quantitative relationships between four biomarker concentrations in urine and dietary intake are shown. CONCLUSION: Design concepts for an analytical strategy are demonstrated, allowing simultaneous quantification of a comprehensive panel of chemically-diverse biomarkers of a wide range of commonly-consumed foods. It is proposed that integration of self-reported dietary recording tools with biomarker approaches will provide more robust assessment of dietary exposure.

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.

Regiochemical Assignment of N-Acylphosphatidylethanolamines (NAPE) by Liquid Chromatography/Electrospray Ionization with Multistage Mass Spectrometry and Its Application to Extracts of Lupin Seeds.

1,2-Diacyl-sn-glycero-3-phospho-N-acyl-ethanolamines (NAPE) are low abundance phospholipids but important constituents of intracellular membranes of plant tissues, responsible for generating bioactive N-acylethanolamine (NAE), which participates in several physiological processes such as regulation of seed germination and protection against pathogenic attacks. From an analytical point of view, the critical aspect of these bioactive lipids lies in the determination of fatty acyl chains located in sn-1/sn-2 position on the glycerol backbone (O-linked), along with the amide-bound (N-linked) fatty acyl chain. Here, the identity and occurrence of NAPE in lipid extracts of lupin seeds (Lupinus luteus L.) was assessed by electrospray ionization in negative ion mode upon reversed-phase liquid chromatography (RPLC-ESI) coupled to mass spectrometry (MS) either at high- (i.e., Orbitrap FTMS) or low- (linear ion trap, LIT) resolution/accuracy. Collisional induced dissociation (CID)-tandem MS and MS3 acquisitions of chemically prepared NAPE allowed to unequivocally recognize the N-linked fatty acyl chain and to establish the diagnostic product ions that were successfully applied to identify NAPE in lipid extracts of yellow lupin seeds. The most abundant NAPE species were those containing N-acyl groups C18:1, C18:2; a minor prevalence was found for C16:0, C18:0, and C18:3, and almost the same acyl chains O-linked on the glycerol backbone in several sn-1/sn-2 combinations were observed. The positional isomers of NAPE species were identified as deprotonated molecules ([M-H]-) at m/z 978.7541 (three isomers 52:3), m/z 980.7694 (two isomers 52:2), m/z 1002.7535 (four isomers 54:5), m/z 1004.7686 (two isomers 54:4), m/z 1006.7837 (two isomers 54:3), and m/z 1008.8026 (single isomer 54:2). The total amount of NAPE in lupin seeds ranged in the interval of 2.00 ± 0.13 mg/g dw, in agreement with other edible legumes. We anticipate our approach to be a robust assessment method potentially applicable to biological extracts containing NAPE species and can provide comprehensive profiles and contents.

Robustness assessment in computer-assisted liquid chromatography procedures based on desirability functions.

A novel approach based on the use of desirability functions is presented for the robustness assessment of liquid chromatographic separations as derived from computer-assisted methods development processes. The approach is based on generally accepted hypothesis that a robust separation procedure will be inert to small random variations of the operational variables, typically encountered in the day-to-day routine analytical practice. This means that peak positions along the chromatograms must keep standstill or move insignificantly when operational variables are not intentionally changed. Thus, the degree of peak positions variation as evaluated from mathematical retention models can be used to assess the robustness of the developed procedures before testing the actual performance experimentally. In the approach proposed, this assessment is obtained by fixing a bilateral partial desirability window around each peak in the simulated chromatogram. The whole chromatogram robustness is characterized by an overall desirability value calculated as the geometric mean of the partial desirability windows evaluation. An added advantage of this approach is that the robustness value calculated is normalized between zero and one and thus, easy to interpret. Thus, when chromatograms are simulated and small random variations are introduced into the operational factors of the model, values for the overall desirability close to one means that the procedure performs robustly. On the contrary, low values for the overall desirability clearly indicated a serious lack of robustness. When used in conjunction with the Pareto optimality approach, as shown here, this robustness assessment strategy allows testing several Pareto front solutions before the final experimental testing which is always needed. In this way, a dramatical reduction of the experimental effort is obtained. Although the approach is theoretically applicable to any chromatographic separation, examples of reversed phase liquid chromatographic procedures are used to show the performance of the proposed methodology.

Bacillus licheniformis peptidoglycan characterization by CZE-MS: Assessment with the benchmark RP-HPLC-MS method.

Peptidoglycan or murein is an essential polymer found in bacterial cell wall. It is a dynamic structure that is continuously remodeled or modified during bacterial cell growth or in presence of cell wall stresses. These modifications are still poorly understood mainly due to the peptidoglycan, which is rather non-soluble, and the difficulties to separate the hydrophilic glycopeptides (muropeptides) by reversed phase liquid chromatography, generated by the enzymatic digestion using mutanolysin, an N-acetyl-muramidase, cleaving the ß1→4 bound between N-acetylglucosamine and N-acetylmuramic acid. Here, we report the use of CZE-MS for an easy and fast screening of muropeptides generated by the action of muramidase on the Bacillus licheniformis cell wall. Electron transfer and CID-MS were also used to unambiguously identify and localize the presence or the absence of amidation and acetylation moieties on muropeptide variants. The reference method to analyse muropeptides by reversed phase chromatography was also tested and the advantages and disadvantages of both methods were evaluated.

Synthetic Peptide Purification via Solid-Phase Extraction with Gradient Elution: A Simple, Economical, Fast, and Efficient Methodology.

A methodology was implemented for purifying peptides in one chromatographic run via solid-phase extraction (SPE), reverse phase mode (RP), and gradient elution, obtaining high-purity products with good yields. Crude peptides were analyzed by reverse phase high performance liquid chromatography and a new mathematical model based on its retention time was developed in order to predict the percentage of organic modifier in which the peptide will elute in RP-SPE. This information was used for designing the elution program of each molecule. It was possible to purify peptides with different physicochemical properties, showing that this method is versatile and requires low solvent consumption, making it the least polluting one. Reverse phase-SPE can easily be routinely implemented. It is an alternative to enrich and purified synthetic or natural molecules.

Determination of 1-Deoxynojirimycin by a developed and validated HPLC-FLD method and assessment of In-vitro antioxidant, α-Amylase and α-Glucosidase inhibitory activity in mulberry varieties from Turkey.

BACKGROUND: Morus alba and Morus nigra leaves which have been widely used as herbal teas in Anatolian region of Turkey, were extracted twice by 50 mM HCI solution, derivatized with 9-fluorenylmethyl chloroformate and analyzed by reversed phase HPLC equipped with a fluorescence detector. HYPOTHESIS/PURPOSE: This study was performed to determine the main antidiabetic active compounds 1-deoxynojirimycin by HPLC method and evaluate the in-vitro antioxidant and antidiabetic activity of ethanol extracts prepared from Morus alba L. and Morus nigra leaves. STUDY DESIGN: A reliable simple, and rapid high-performance liquid chromatographic (HPLC) method for the determination of 1-deoxynojirimycin in M. alba L. and M. nigra leaves with fluorimetric detection after pre-column derivatization with 9-fluorenylmethyl chloroformate was developed. In addition, the chemical composition of ethanol extract of mulberry leaves was analyzed with GC-MS. METHODS: Separation and quantitation were performed on C18, 250 × 4.6 mm, 5 µm analytical column. Mobile phase consisted of acetonitrile and 0.1% acetic acid solution (1:1, v/v) was performed applied to the column 1.0 ml/min flow rate at 26 °C. Potential antioxidant activity of ethanol extract of different mulberry varieties were evaluated by DPPH, and ABTS radical scavenging assay as well as total phenol and flavonoid content were determined. In addition, α-amylase and α-glucosidase activity was determined by 96-well plate method to evaluate the probable antidiabetic potential use of Turkish mulberry leaves. RESULTS: The isocratic HPLC method showed excellent correlation coefficient (r2 = 0.9985) between 0.3 and 30 µg/ml calibration points. The method was specific and sensitive with detection and quantification limits of 1.07 and 3.27 ng/ml, respectively. Intraday and interday method precision (n = 5) were < 7.3 (RSD%). Intraday and interday method accuracy (n = 5) were between 3.77 and (-8.35) (RE%). The average method recovery (n = 3) was 102.5%. The results showed that the content of 1-deoxynojirimycin in leaves of Morus alba L. was 0.103% (n = 3), and in leaves of M. nigra L. was 0.102%. 2-hexadecen-1-ol, oleamide, 2-propenoic acid, and cyclododecane were identified as the major compounds by GC-MS in the ethanol extract of mulberry leaves. CONCLUSION: The obtained robustness values from emission and excitation detection, mobile phase ingredients and flow rates changes showed that method was very strong. This work contributes to the knowledge of antioxidant and antidiabetic properties of Morus species, thus may be provide useful data in evaluation of food products and pharmaceutical preparations produced from Morus species.