Percentage Of Organic Modifier Research Articles

Mixed-mode separation of antisense oligonucleotides using a single column with complementary anion-exchange and hydrophobic interaction chromatography approaches

The current study investigates the use of mixed-mode chromatography as a combination of anion-exchange (AEX) and hydrophobic interaction chromatography (HIC) for the analysis and purification of single-stranded antisense oligonucleotides with stereo-controlled phosphorothioate inter- nucleotide linkages.Initially a Scherzo-SS-C18 trimodal stationary phase with reversed-phase/AEX/ cation-exchange (CEX) functionalities is systematically evaluated to reveal the presence of U-shaped retention composed of two retention modes namely AEX and HIC, where the latter was also observed on related trimodal Scherzo SM and SW analogues. For the first time, retention and separation of deprotected oligonucleotides was described on a single mixed-mode column using a combination of AEX and HIC. This methodology was later applied to an alternative reversed-phase/AEX support, Newcrom BH, displaying similar retention trends under dual salt / organic modifier gradients. The merit of the method was assessed on the basis of separations between a phosphodiester (PO) impurity and phosphorothioate (PS) target for an assortment of selected 2’-O-methoxyethyl 18- to 20-mer single-stranded antisense oligonucleotides. Various parameters were evaluated mostly under HIC conditions including organic modifier percentage, type of salt, temperature, pH and type of buffer in the mobile phase. Retention of the oligonucleotides was significantly affected by the acetonitrile composition and the type of salt ((NH4)2SO4, NaBr, NaCl) where the latter NaCl also afforded resolution between the PS target and closely eluting PO impurities. Small changes in pH between 6.5 and 7 using MES and TRIS respectively demonstrated notable differences in retention and resolution. The optimized methods were compared against a range of traditional supports and applied to various mixed-mode analogues, possessing embedded amino acid, complex forming weak cation-exchange and terminal strong cation-exchange functionalities. The mixed-mode supports displayed HIC retention and better resolution between PS target and PO impurity was evident with a more focused 5% acetonitrile gradient over its 10% counterpart. Overall, throughout the study AEX and HIC demonstrated comparable resolution trends.Finally, the optimized HIC method was applied to a selected 18-mer antisense oligonucleotide at semi-preparative scale using the Newcrom BH column, affording purities of 60-80% and recoveries of 52-76%. Although HIC does not provide better separations between PS and PO than AEX, it opens possibilities to operate under non-denaturing conditions and allows purification of samples containing high salt content, as a standalone method or post AEX without a prior desalting step, which can result in 20-30% sample loss.

Stability Indicating RP-HPLC Method for Posaconazole Assay Using QbD Approach.

The development and optimization of analytical techniques utilizing the quality by design methodology is essential in the pharmaceutical industry. This approach helps in the direction of identifying and optimizing critical parameters during drug development and assesses their impact on key quality attributes. A stability-indicating RP-HPLC method was developed for quantifying posaconazole in bulk and tablet forms utilizing the quality-by-design approach. The Box-Behnken design optimized buffer flow rate, organic modifier percentage, and temperature, affecting retention time, theoretical plate count, and symmetry factor. Chromatography was performed on a Symmetry C18 column with a mobile phase of 0.01N potassium dihydrogen phosphate and acetonitrile (57.4:42.6% v/v), at a flow rate of 1.11 mL/min, detection at 220 nm, and column temperature of 30°C. Posaconazole’s retention time was 3.663 minutes, with precise, accurate, and linear signals (R² = 0.999). The method is simple, fast, cost-effective, and ideal for routine quality control.

Study of Different Chiral Columns for the Enantiomeric Separation of Azoles Using Supercritical Fluid Chromatography

The enantiomeric separation of antifungal compounds is an arduous task in pharmaceutical and biomedical fields due to the different properties that each diastereoisomer presents. The enantioseparation of a group of fungicides (sulconazole, bifonazole, triadimefon and triadimenol) using supercritical fluid chromatography was achieved in this work. For this goal, four different chiral columns based on polysaccharide derivatives, as well as the effect of different chromatographic parameters such as temperature, type and percentage of organic modifier (methanol, ethanol and isopropanol), were thoroughly investigated. The inversion of the elution order of enantiomers as a result of a change in the stationary phase or organic modifier was also evaluated by employing a circular dichroism detector. The best separation conditions, in terms of the enantioresolution and analysis time, were obtained with the Lux® Cellulose-2 column using isopropanol as the organic modifier.

Chiral LC method development: Stereo-selective separation, characterization, and determination of cabotegravir and related RS, RR, and SS isomeric impurities on coated cellulose-based chiral stationary phase by HILIC-LC and LC-MS

The first, sensitive, and rapid chiral method was developed for enatioseperations and determination of cabotegravir and its enantiomeric impurities by using HPLC and LC-MS. Cabotegravir is an antiretroviral medication used for the treatment of HIV/AIDS approved by the food and drugs administration (FDA) in the year 2021. The cabotegravir chiral separation was achieved on the coated cellulose-tris (4-chloro-3-methyl phenyl carbamate) (CHIRALCEL OX-3R) column in HILIC mode and the total run time is less than 15 min. The effects of mobile phase composition, elution mode, and percentage of organic modifier as well as the effect of mobile phase-additives and column temperature were investigated on selectivity, resolution, and peak symmetry. The mobile phase consisted of acetonitrile and water with 0.1% (v/v) addition of formic acid additive with the flow rate of 1 mLmin−1. UV detection was carried out at 220 nm. The calibration curves of cabotegravir and its enantiomers were linear over the concentration range of 0.04–1.125 µgmL−1. The limits of detection and quantification for cabotegravir and its enantiomer (RS isomer) were ≤ 0.02 and ≤ 0.06, and the RR and SS-isomers limits were ≤ 0.02 and 0.03 µgmL−1 respectively. It was demonstrated that the proposed method is selective, precise, and robust. Finally, the validated method was applied for the determination and identification of cabotegravir and its chiral enantiomers in the bulk drugs by using HPLC and LC-MS techniques.

UHPLC Enantiomer Resolution for the ɑ/β-Adrenoceptor Antagonist R/S-Carvedilol and Its Major Active Metabolites on Chiralpak IB N-5.

Carvedilol (CAR), a racemic lipophilic aryloxy propanolamine, acts as a selective α1-adrenoreceptor antagonist and a nonselective β-adrenoreceptor antagonist. CAR metabolism mainly produces three active metabolites: desmethyl carvedilol (DMC), 4′-hydroxy carvedilol (4′OHC) and 5′-hydroxy carvedilol (5′OHC). The oxidative S-(-)-metabolites contribute to the β-antagonistic effect, yet not to the α-antagonistic effect to be observed after drug dosage. Therefore, the three β-adrenoceptor blocking metabolites, which are structurally closely related to the parent CAR, are included into the development of a bioanalytical quantitative method for all major active species relevant with respect to adrenoceptor-blockade. Because of the given pharmacological profile, resolution of the enantiomers of carvedilol, of 4′- and 5′-hydroxy carvedilol as well as of DMC, is mandatory. The current study aims to determine the response surface for the enantiomer separation of the parent CAR as well as the major metabolites on a suitable chiral stationary phase. Design of experiment approach (DoE) was utilized in an initial screening phase followed by central-composite design for delimitation of the response surface for resolution of the four enantiomeric pairs in least run time. The impact of chromatographic variables (composition and percentage of organic modifier(s), buffer type, buffer pH, flow rate) on critical peaks resolution and adjusted retention time was evaluated, in order to select the most significant critical quality attributes. On this basis, a robust UHPLC-UV method was developed and optimized for the simultaneous, enantioselective determination of CAR along with its major active metabolites (4′OHC, 5′OHC, and DMC) on Chiralpak IBN-5. The optimized UHPLC-UV method (which includes metoprolol as the internal standard) was validated according to the ICH M10 guidelines for bioanalytical methods and proven to be linear, precise, accurate, and robust. The validated assay was applied to plasma samples from cardiovascular patients treated with rac-CAR (blood randomly drawn at different times after oral CAR intake). In order to provide more insight into the mechanism of the enantiomer separation of CAR and its metabolites on the CSP, docking experiments were performed. Molecular simulation studies suggest the chiral recognition to be mainly due to different binding poses of enantiomers of the same compound.

Predicting skin permeability of pharmaceutical and cosmetic compounds using retention on octadecyl, cholesterol-bonded and immobilized artificial membrane columns

In this study, the retention on three types of columns, an immobilized artificial membrane (IAM), a cholesterol-bonded and an octadecyl (C18) column, was applied for the prediction of skin permeability. The first two columns are biomimicking ones, which have certain components of the skin bound to the stationary phase, and were applied in HPLC, while the sub-2 µm C18 column was studied in UHPLC because of its fast features. Fifty-eight compounds were analyzed applying different mobile-phase compositions, with varying percentages of organic modifier on every column, to extrapolate the retention factor to a theoretically purely aqueous mobile phase (log kw). The retention factors, along with two sets of theoretical molecular descriptors, were used to model the skin permeability coefficient (log Kp) using multiple linear regression (MLR) and partial least squares (PLS) regression modelling. Although the retention factors (log k) on the IAM column showed a better correlation with the skin permeability, the overall best model was obtained by applying a stepwise MLR approach on the UHPLC parameters combined with some theoretical descriptors. This model showed a good fit, and on top has potential to accurately predict skin permeability values. Furthermore, the UHPLC method has the advantage of being fast and can thus be classified as a high-throughput approach.

Hydrophilic Interaction Chromatography with Sulfobetaine Zwitterionic Polymer-Bonded Stationary Phases

A rapid method for simultaneous determination of two statin drugs based on zwitterionic chromatography (ZIC) had been developed and validated. The development method included examining the effects of chromatographic conditions, including the percentage of organic modifier, pH values, ionic strength of the acetate buffer, and the predominant retention mechanismand#39;s experimental determination. Separation developed by two zwitterionic stationary phases (100 mm and#215; 4.6 mm I.D., 3.5and#181;). The influence of various spacer lengths was being used as an examination tool on atorvastatin and rosuvastatin retention behaviours. Two zwitterionic stationary phases and a mobile phase consisting of acetonitrile and acetate (pH = 4.75, 40 mM) in a ratio of 80:20 V/V were used to achieve optimum chromatographic conditions. The methods had been validated for linearity, accuracy, and precision. This validation shows that the ZIC-HILIC methods proposed were sufficient for quantification analysis of atorvastatin and rosuvastatin. Quantifications were achieved with U.V. detection at 240 nm over the concentration range of 0.1–7.0 μg mL-1 for atorvastatin and rosuvastatin, respectively.

Simultaneous detection of hazardous skin whitening agents in Indian cosmetic products using a green chromatographic technique

The present work mainly highlights the simultaneous detection of four skin whitening agents i.e. hydroquinone (HQ), resorcinol (RS), catechol (CC) and 3,3′-dichlorobenzidine (DCB) in facial creams and body lotion. Among these, the first three are positional isomers of dihydroxybenzene so simultaneous separation is difficult with the conventional reverse-phase high-performance liquid chromatographic technique (RP-HPLC). The selected skin whitening agents were detected in facial cream and body lotion using micellar liquid chromatography coupled to a photodiode array detector (MLC-PDA). In the present study, optimization of the method was accomplished using response surface methodology (RSM) with central composite design (CCD). The second-order polynomial model for predicting the optimal chromatographic run time was evaluated by the analysis of variance (ANOVA) and 3D response surface plots for the interactions between three variables were constructed. Three experimental parameters which were chosen as independent variables were; surfactant concentration (SDS), the volume percentage of organic modifier (OM) and pH of the mobile phase. The second-order polynomial model for predicting the optimal chromatographic run time was evaluated by the analysis of variance (ANOVA) and 3D response surface plots for the interactions between three variables were constructed. Three experimental parameters which were chosen as independent variables were; surfactant concentration (SDS), percentage of organic modifier (OM) and pH of the mobile phase. The second-order polynomial model for predicting the optimal chromatographic run time was evaluated by the analysis of variance (ANOVA) and 3D response surface plots for the interactions between three variables were constructed. Three experimental parameters which were chosen as independent variables were; surfactant concentration (SDS), percentage of organic modifier (OM) and pH of the mobile phase. The optimized mobile phase was 0.15 M SDS and 7% 1-butanol, buffered at pH 7 with 0.01 M NaH2PO4. The chromatographic run time for simultaneous determination of selected analytes was 7.5 min. The correlation coefficient (r2) values were satisfactory between 0.998–0.999 over the linear concentration range. Limits of detection (LODs) and the limits of quantification (LOQs) for the four skin whitening agents were in the range of 0.05–0.07 μg/mg and 0.11–0.14 μg/mg, respectively. Trueness (98.4–102.7%) and precision (< 4.3%) were acceptable. The developed method was fast, cost-effective, and green which could easily analyze complex matrices (facial creams, body lotion) without any pretreatment other than filtration. The results indicated that the MLC-PDA method proved to be more suitable for the simultaneous separation of selected positional isomers.

Optimization of a Ligand Exchange Chromatography Method for the Enantioselective Separation of Levofloxacin and Its Chiral Impurity.

Levofloxacin is a third-generation fluoroquinolone that has several advantages over its (R) ofloxacin isomer. It is used to treat different types of infection, including urinary infection and prostatitis. A new HPLC method for the enantioselective separation of levofloxacin and its chiral impurity was developed and validated to improve the separation of the enantiomers of levofloxacin [impurity(R) and active principle (S)] by increasing the value of the resolution between the eutomer and the distomer. Chromatographic separation was performed on a Prodigy ODS -2, 5 µm 4.6 × 150 mm column, with a gradient of buffer solution and methanol (80:20, v/v). A Box-Behnken design was considered when optimizing the enantioseparation involving the effects of many factors such as the concentration of d-phenylalanine, the pH of the buffer, the percentage of organic modifier in the mobile phase, the flow rate, the temperature of the column, and the type of column. Chiral separation was achieved with an optimal resolution of 3.8. The method was successfully validated following the International Conference on Harmonization Q2 (R1) guideline, fulfilling the acceptance criteria for selectivity [no interference in the retention time of (S) levofloxacin and (R) levofloxacin], linearity (r ≥0.999 in the range 1.25-3.75 µg/mL for all enantiomers), and precision (RSD <2%). Accuracy was assessed by the application of the analytical method to an analyte of known purity, providing evidence for the usefulness of this monitoring system. The method was successfully used for the determination of levofloxacin impurity in raw material and pharmaceutical dosage forms. The following method is accurate and robust to quantify and characterize the presence of levofloxacin impurity in raw material for pharmaceutical compounds.

Sulfated Polyborate, a Novel Buffer for Low pH Mobile Phase on a Nonend Capped Stationary Phase in Reverse Phase Liquid Chromatography

Background: Sulfated Polyborate, a novel inorganic material primarily designed as a catalyst, has shown properties such as high solubility in organic solvents, low U.V. cut-off, and pKa ≈2.0, which suggests its potential as a mobile phase buffer for reverse-phase liquid chromatography. Objective: This study aims to substantiate the role of Sulfated Polyborate as mobile phase buffer for reverse-phase liquid chromatographic analysis of basic drugs with high pKa values viz. Bisoprolol fumarate, Timolol maleate, Verapamil hydrochloride, and Carvedilol. Methods: Solubilities, U.V. cut-offs, and pKa of Sulfated Polyborate were first experimentally confirmed. The behaviour of Sulfated Polyborate as mobile phase buffer at pH 3.0 was ascertained by varying the buffer concentration, flow rates, and percent organic modifier for elution of the four basic drugs on a non-end capped octyl silyl (C8) column. Similarly, the study was performed with KH2PO4 as a reference buffer. The column performance and conductometric measurements ascertained the impact of Sulfated Polyborate on the stationary phase. Results: Sulfated Polyborate and KH2PO4 buffers showed correlation coefficients of 0.99 and 1.00 for analyte retention factors for variation of buffer concentration and organic modifier composition, respectively. Peak symmetries and the number of theoretical plates were improved from &gt; 2.0 to &lt; 2.0 and ≈1000 to ≈3000, respectively, for variation in buffer concentrations. Similar Van Deemter plots indicated equivalency of Sulfated Polyborate and KH2PO4 buffers. The column performance and conductometric measurements depicted no adsorption on the stationary phase. Conclusion: The present study demonstrates Sulfated Polyborate as a novel buffer for analytes with higher pKa on reverse-phase liquid chromatography.

Development and Validation of a Ultra Flow Liquid Chromatography Method for the Assay of Boceprevir Using a Quality-by-Design Approach

Quality by design guided. The assay method of Boceprevir is developed in accordance with ICH Q8(R2) guideline with due validation. .In this process, the Target analytical profile (TAP) of the drug was set and critical method parameters (CMP) were investigated by systematic risk assessment experimentation to control critical Quality Attributes (CQA). In this, A Cause Effect Risk Assessment Matrix with Control-Noise-Experiment (CNX) is used for identifying the high-risk variables i.e Percentage of Organic Modifier (% methanol), pH of the Buffer and flow rate of the mobile phase. The surface response methodology was applied to optimize the critical method parameters (CMP) as well as Critical Quality Attributes (CQA) to find out the Design space of the method. The Optimum assay method condition was mobile phase Acetate Buffer (50mM) pH 5.4: Methanol (11:89), Flow rate: 0.9 ml/min, Lambda Max: 207. The separation was achieved in the Eclip Plus C-18 column (250 × 4.6 mm, 5μm) at ambient temperature. The retention time of Boceprevir was found to be 4.2 min. The method evaluation was performed according to the (Q2R1) ICH guideline.

Development of Ion pair Chromatography Method for Assay of Telaprevir by Response Surface Methodology

Response surface methodology approach has been utilized for the assay of Telaprevir in pure and formulation using ion-pair chromatography. In this, A risk assessment approach (Control-Noise-Experiment) has been used for identifying the risk factors, i.e. Percentage of Organic Modifier (% Acetonitrile), Buffer’s pH and flow rate of the method. The Central Composite design was applied to optimize the critical method parameters (CMPs) and to find out the Design space (DS) of the method. The coefficient of correlation (R2),%CV and Lack of fit are utilized for the evaluation of method responses (Retention time and Asymmetric factor Evaluation of model is justified by two diagnostic plots (normal probability plot of residuals and plot of residuals vs predicted values). The mobile phase is Acetate Buffer (20mM) pH 4.4: Acetonitrile (35:65) with 0.9 ml/min of Flow rate. The separation has taken placed in the Eclip Plus C-18 column (250 × 4.6 mm, 5μm) at 268 nm. The retention time of Boceprevir was found to be 4.6 min. The validation of the optimized method has performed according to ICH guideline. The method has been successfully used for routine analysis of the Telaprevir throughout the life cycle of the product.

Ultra-high performance supercritical fluid chromatography coupled to tandem mass spectrometry for antidoping analyses: Assessment of the inter-laboratory reproducibility with urine samples.

The aim of this study was to assess the interlaboratory reproducibility of ultra-high performance supercritical fluid chromatography coupled with tandem mass spectrometry method for routine antidoping analyses. To do so, a set of 21 doping agents, spiked in urine and analyzed after dilute and shoot treatment, was used to assess the variability of their retention times between four different laboratories, all equipped with the same chromatographic system and with the same ultra-high performance supercritical fluid chromatography stationary phase chemistry. The average relative standard deviations (RSD%) demonstrated a good reproducibility of the retention times for 19 out of 21 analytes, with RSD% values below 3.0%. Only for two substances, namely fenbutrazate and niketamide, the retention was not repeatable between laboratories, with RSD% of approximately 15% in both cases. This behaviour was associated with (a) the low organic modifier percentage (around 2-4%) in the mobile phase at the corresponding retention times, and (b) the influence of the system volume on poorly retained analytes. An analysis on seven "blind" urines was subsequently carried out in the same four laboratories. In these blind samples, either one, two, or none of the 21 doping agents previously analyzed were present at an unknown concentration. Each laboratory had to perform the identification of the compounds in the samples and estimate their concentrations. All laboratories assigned all target analytes correctly in all blind urine samples and provide a comparable estimation of their concentrations.

Investigating the use of unconventional temperatures in supercritical fluid chromatography

The use of unorthodox temperatures, ranging from −5 °C up to 80 °C, have been thoroughly investigated in supercritical fluid chromatography. To this purpose, an initial evaluation of the kinetic and thermodynamic performance has been made with a set of 4 analytes eluting at different percentages of organic co-solvent in the mobile phase (3%–10% - 45%–80%). The van Deemter plots have demonstrated how, at low organic modifier presence, the use of low temperatures did not necessarily translate into worse performance, while high temperatures could pose more issues due to the poor handling of the super/subcritical mobile phase by the chromatographic system. With important percentages of co-solvent, however, high temperatures were fundamental in ensuring better profiles of the van Deemter plots, compared to low temperatures. Pressure plots have demonstrated that gradients reaching elevated percentages of organic modifiers can also be used on stationary phases packed with sub 2 μm silica particles if high temperatures are employed. The thermodynamic evaluation, made via the analysis of van’t Hoff plots, indicates the presence of three retention behaviors happening in UHPSFC when switching from high to low temperatures, depending on the co-solvent percentage needed to elute one analyte. Finally, an assessment of the stationary phase stability at high temperatures was performed: the retention times variabilities recorded were minimal (RSD < 2.5%), as well as the peak widths and inlet column pressures were somewhat constant throughout the analyses.In the second part of this study, a focus on potential applications benefiting from such unconventional temperatures has been made. A series of challenging analytes have experienced better chromatographic resolution at either high or low temperatures, providing therefore a potentially interesting tool to analysts during the chromatographic method development process. In conclusion, the UV sensitivity at different temperatures was also taken into consideration, with no significant impact on the quality of the UV signal under any condition.

Stress testing of crisaborole by a novel stability indicating RP-HPLC method

An isocratic reverse phase high performance liquid chromatographic method has been developed for the determination of crisaborole (CRB), a novel non-steroidal topical phosphodiesterase-4-inhibitor. The analyte was separated on an Agilent eclipse XDB-C18 (3.0 × 150 mm, 3.5 µm) analytical column using water: acetonitrile (30: 70 v/v) as the mobile phase at a flow rate of 0.75 ml/min under ambient temperature and PDA detection at 254 nm. Added to this, several validation parameters were exercised on the developed HPLC method as per ICH guidelines. Method variables, viz., mobile phase flow rate (0.75 ± 0.1 ml/min) and percentage of organic modifier in mobile phase (70 ± 5%) were studied by a central composite design for testing the robustness of the proposed method. The method was found to be linear in the concentration range of 2–100 µg/ml (r2 = 0.999) and precise (RSD ˂ 2%) satisfying regulatory criteria. The method was found to be robust and ambiguity of worst result for certain responses still are acceptable and likely to occur due to the effect of extreme experimental conditions. Stress testing of CRB was carried out under acid, base, thermal, photolysis, peroxide and neutral conditions. Separation of the forced degradation products from the main analyte was accomplished using a gradient elution of the mobile phase consisting acetonitrile from 0 to 100% in water at a flow rate of 1.2 ml/min.

Usual, unusual and unbelievable retention behavior in achiral supercritical fluid chromatography: Review and discussion

Retention rules are well known in liquid chromatography. For the mobile phase composition, retention decreases when adding organic solvent to water for reversed-phase or increasing water proportion for hydrophilic interaction liquid chromatography, and a decrease in temperature usually increases retention. For supercritical fluids, the fluid density, which is related to temperature and column back-pressure, is significant for neat CO2 and with low percentages of organic modifiers, i.e. with compressible mobile phases. The increase in the modifier percentage reduces the fluid compressibility, leading to retention behaviors close to those observed with liquid mobile phases, for instance for temperature changes. Moreover, adsorption of carbon dioxide or modifiers modify the solutes/stationary phase interactions, further complicating the understanding of the observed retention changes, either with low amount of modifier, or with specific modifiers.Besides, the polar and nonpolar stationary phases (SPs) do not always behave identically, depending on physico-chemical properties. Silica, amino or ethyl-pyridine polar phases display mostly identical behavior for classical differences of compounds of different polarity, but can provide different retention order for more subtle differences, such as the position of polar groups. Moreover, the nature of the silica, inorganic or hybrid, or the additional charges onto the silica surface can also lead to different results.Even if the C18-bonded phases are not as popular as polar SPs, the non-polar SPs provide very high separation performances for suited compounds, i.e. for non-polar compounds, which are perfectly solubilized by supercritical fluids. Recently, unusual retention behaviors were observed with some specific C18-bonded phases, which display polar interactions in addition to dispersion interactions.Whatever the SPs used, supercritical fluids appear to favor specific effects that are not observed with liquid mobile phases that are more uniform in terms of physico-chemical properties.The objective of this paper is to describe different separation behaviors observed in SFC, to improve the general understanding of the specificities of the association of supercritical fluids and varied SPs.

Determination of atmospheric particle-bound polycyclic aromatic hydrocarbons using subcritical water extraction coupled with membrane microextraction

A green analytical methodology for the determination of 16 priority polycyclic aromatic hydrocarbons (PAHs) and 8 related compounds in air particulate matter was developed and validated. The method was based on pressurized hot water extraction (PHWE) followed by miniaturized membrane-assisted solvent extraction (MASE) and programmed temperature vaporization-gas chromatography-ion trap tandem mass spectrometry detection (PTV-GC–MS/MS). The parameters studied for PHWE were percentage of organic modifier (25% MeOH), temperature (200 °C) and static extraction time (5 min). For MASE, extraction temperature (30 °C), time (90 min) and effect of an organic modifier were also optimized. The matrix effect was evaluated and compensated using deuterated labelled standards as surrogates for the quantitation of the target compounds. The analytical performance of the method was satisfactory: relative recoveries varied between 78 and 118% and repeatability and intermediate precision were <22% for all compounds. The Method Quantitation Limits (MQL) ranged from 0.9 (TPY) to 75.6 pg m−3 (NAP). Satisfactory results for accuracy and traceability were evidenced by the analysis of a reference material (SRM 1649b) and comparison with previously published methods. The greenness score was estimated and compared with other techniques widely used for the analysis of PAHs in particulate matter, having a lower relative environmental impact.

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.

Six Sigma quality approach for HPLC-UV method optimization

We report a robust HPLC-UV method for ascorbic acid (AS), paracetamol (PA) and guaifenesin (GU) based on response surface/tolerance analysis approach to meet Six Sigma quality of the performance of a chromatographic method. Our goals were the bringing specifications into the optimizations and achieving robustness during the development phase. The critical method attributes (CMAs) were resolution between the peak pairs (Rs), the analysis time(T), tailing factor (Tf), number of theoretical plates (N), and back pressure(P).The critical method parameters (CMPs) included a percentage of organic modifier, a flow rate of the mobile phase, and a temperature of column oven. The separation was performed on a CN column, using an isocratic mode with a mixture of 50 mM potassium dihydrogen phosphate buffer adjusted with ortho-phosphoric acid to pH 2.2 as the aqueous component of the mobile phase and methanol in the ratio 85.9:14.1 (v/v) at 1 ml/min flow rate, with UV detector at 275 nm and a column temperature of 45 °C. The method was validated using the accuracy profiles. Finally, the method was applied to pharmaceutical dosage form.

Polymeric stationary phases based on poly(butylene terephthalate) and poly(4-vinylpirydine) in the analysis of polyphenols using supercritical fluid chromatography. Application to bee pollen

Two new polymer-based stationary phases; DCpak PBT (poly(butylene terephthalate)) and DCpak P4VP (poly(4-vinylpirydine)) were evaluated for the analysis of polyphenols using supercritical fluid chromatography (SFC). The compounds studied included phenolic acids and flavonoids. The different variables that influence the chromatographic separation, such as type and percentage of organic modifier, additive, pressure and temperature were examined. Using the DCpak P4VP column the retention was exceptionally high, obtaining better results with the DCpak PBT column. The separation of nine polyphenols was achieved using a gradient of modifier (methanol with 0.1% trifluoroacetic acid) from 5 to 50%, a pressure of 150 bar, a temperature of 35 °C and a flow-rate of 2 mL/min. The use of additives was necessary in order to obtain good peak shapes and efficiencies, achieving the best results with trifluoroacetic acid. LODs and LOQs values were lower than 5 μg/mL in all the cases; meanwhile, the %RSD values for method repeatability and inter-day reproducibility were lower than 3% and 10% respectively. Finally, the proposed method was successfully applied to the analysis of polyphenols in commercial bee pollen; four compounds, namely cinnamic acid, p-coumaric acid, catechin and quercetin were identified and quantified.