Core-shell Column Research Articles

The best structures of LC columns—A theoretical perspective

The largest peak capacity (n) that LC analysis can generate in isocratic or gradient elution analysis of a given sample in a given time at a given pressure is proportional to the quality factor (qmax) of its column structure. In this study, the multi-channel structures with open pseudo-planar channels (OPPC) and open circular channels (OCC) where compared with PC2 – a typical core-shell column packed with 2 μm particles. These columns have qmax of 1.27, 1.17 and 0.41, respectively. The former two qmax are the highest among all known column structures – about 3 times higher than qmax of PC2. This means that the OPPC and OCC can generate about 3 times higher n compared to what a PC2 can in the same analysis time (tanal) at the same pressure, or they require about 81 times shorter tanal (81 is the 4th power of 3) to generate the same n as a PC2 can at the same pressure. However, while PC2 is a commercially available column, there are substantial challenges in manufacturing the OPPC and OCC that can compete with PC2 in practical applications. In order to be competitive with PC2, the OPPC and OCC should have sub-1μm characteristic dimensions (e.g., the inter-pillar distance, g, in OPPC-based pillar array columns, internal diameters of OCC). Thus, in order to compete with PC2 in one scenario, an OPPC requires g ≤ 0.14 μm. Additionally, to be competitive with PC2, OPPC and OCC should be able to sustain the same high pressure. Highlighting the challenges of their design and manufacturing might help to develop the manufacturable columns substantially superior to the packed ones.

Novel green HPTLC and organic solvent-free micellar LC methods for the simultaneous determination of Ozenoxacin and benzoic acid; greenness assessment and applications

Ozenoxacin (OZ) is a novel synthetic quinolone antimicrobial agent that has been recently approved by US-FDA for the treatment of impetigo in combination with its preservative, benzoic acid (BA). Meanwhile, implementing greener chromatography has been a recent goal of a great ecological importance. In the proposed research, two novel green methods have been validated for the simultaneous determination of OZ and BA in skin creams for the first time. An organic-solvent free mixed-micellar HPLC technique was developed using RP-C8 core-shell column with a mobile phase consisting of 0.01 M Brij-35, 0.15 M sodium dodecyl sulfate, and 0.02 M ammonium acetate in water, adjusted to pH 4.5. The mobile phase flow rate was 1 mL min−1 and detecting analytes in short retention times (3.4 and 4.7 min for BA and OZ, respectively) at wavelength 235 nm. The working concentration ranges were 1–10 and 10–100 μg mL−1 for BA and OZ, respectively. Another eco-friendly high-performance thin-layer chromatography (HPTLC) method was designed using HPTLC silica-gel 60 F254 plates as a stationary phase and ethyl acetate–ethanol–ammonia (6:4:2, v/v/v) as mobile phase. The detection was also performed at λmax = 235 nm. The method was found linear across concentration range of 0.5–1.8 and 0.05–0.18 μg/band for OZ and BA, respectively which gave symmetric, compact band with Rf values of 0.21 and 0.69 for OZ and BA, respectively. The proposed methods were applied successfully in their combined marketed pharmaceutical cream. The proposed methods were typically based on safe biodegradable reagents, and their ecological superiority was proven using recent greenness metrics.

B-182 Advantage of Core-Shell Particle Morphology Over Fully Porous HPLC Column for the Analysis of Vitamin K Compounds by LC-MS/MS

Abstract Background Vitamin K’s are a group of structurally similar, fat-soluble compounds. There are two natural forms K1 (Phylloquinone) and K2 (Menaquinone), where the later has short and longer chain homologues called Vitamin K2-MK-4 and Vitamin K2-MK-7 respectively. Due to the extremely hydrophobic nature and poor ionization characteristics of these compounds, LC-MS/MS analysis at low concentrations becomes challenging. In this communication we make parallel comparisons of three HPLC columns packed with fully porous and core-shell particles to showcase the superiority of core-shell columns for sensitive detection of vitamin K analytes by LC-MS/MS. Method We compared fully porous (Gemini and Luna, Phenomenex, Torrance, CA) and core-shell (Kinetex EVO, Phenomenex) C18 columns for this study. All columns evaluated were 50 × 2.1 mm with a 5 μm particle size to maintain low back pressure for the viscous organic modifier utilized. Deionized water and a combination of isopropanol and acetonitrile (1:1; v/v) were used as mobile phase A and B respectively for chromatographic separation. An Agilent 1290 Infinity series LC system was employed with a SCIEX 6500 QTRAP mass spectrometer under APCI for MS detection. Analyst software 1.7.2 used for data analysis. Results The LC-MS/MS method had a 4-minute runtime for chromatographic separation of the analytes including 1.5 min of equilibration time. The comparison data revealed the Gemini C18 column shows some higher selectivity and resolution values, whereas both EVO and Luna C18 columns were somewhat comparable. However, the core-shell EVO C18 column displayed much higher efficiency with narrower and compressed peak widths, which dramatically increased the peak height for all 3 analytes exhibiting the highest signal/noise ratio. Conclusion The core-shell Kinetex 5 μm, EVO C18 column with its unique organo-silica grafting shows much higher efficiency and sensitive detection capability for vitamin K analysis by LC-MS/MS compared to that of conventional fully porous silica particles.

Rapid Analysis of Four Alkaloids in Uncaria rhynchophylla by Core-Shell Column HPLC and Quantitative Analysis of Multi-Components by Single Marker (QAMS)

As a traditional herbal medicine, the major alkaloids in Uncaria rhynchophylla have been proven to have blood pressure-lowering and sedative effects. It is essential to develop an effective method for the determination of the major alkaloids in U. rhynchophylla. In this research, a rapid quantitative analysis involving multi-components analysis by a single marker strategy coupled with core-shell column HPLC was adopted to analyse four alkaloids (corynoxeine, isocorynoxeine, isorhynchophylline, rhynchophylline) in U. rhynchophylla. Isorhynchophylline was selected as the internal reference substance, the content of which was determined by the traditional external standard method. Relative correction factors (RCF) between isorhynchophylline and the other three alkaloids were calculated respectively. The results showed that the QAMS method had good robustness under different HPLC instruments. Nineteen batches of U. rhynchophylla were tested. No significant difference was observed between the results by QAMS and EMS (Correlation coefficient > 0.99, p > 0.05). The QAMS method could be employed as a rapid, effective technique for the quality control of U. rhynchophylla.

Mixed-mode chromatography of mixed functionalized analytes as the homologues of benzalkonium chloride. Application to pharmaceutical formulations

In this work, a retention behavior based on mixed-mode reversed-phase (RP)/hydrophilic interaction liquid chromatography (HILIC) was observed for benzalkonium chloride (BAK) using a core-shell column functionalized with biphenyl groups. Although in the literature, the U-shaped retention was reported for polar compounds in mixed functionalized phases, in the present work, the behavior was dependent upon the chemical structure of the analyte with mixed functionality (ammonium group, a benzyl group and an alkyl chain) and on the high selectivity of the chromatographic column. The bimodal retention was observed for the four BAK homologues using a content of acetonitrile from 65 to 95% in the mobile phase. The data were adjusted to polynomial equations which allow for modeling and predicting the U-shaped retention. The salt concentration (50 and 100 mM), anion (formate and acetate) and cation (ammonium and triethylammonium) of the salt, pH (4 and 5) in the mobile phase were studied in order to understand their influence on the two retention modes. Significant electrostatic interactions were involved in the two retention modes, especially with a content of acetonitrile higher that 90%. Linear relationships between the retention factors of the four homologues were found in a wide range of %acetonitrile when the salt and triethylamine concentration, pH and nature of salt were changed. The differences found on the retention of the homologues, when increasing the alkyl chain length, were more significant in the RP mode due to predominant hydrophobic interactions. A pH decrease and a salt concentration increase caused a retention decrease for both modes. A decrease on of the retention was observed when acetate anion was replaced by formate anion. The different order of the polynomial equations according to the used mobile phase confirmed its relevant role in the interactions with the analytes and stationary phase. A mobile phase was selected (85% acetonitrile, pH 4 and 100 mM ammonium formate) for the BAK determination in cutaneous, otic and ophthalmic formulations with different active pharmaceutical ingredients and excipients. Low sample volume (500 μL) and short analysis time (<5 min) were some of the advantages of the proposed method. In addition, good analytical performance (R2 > 0.999, % RSD <4.5% for intra-day precision and <5.8% for inter-day precision, and recoveries in the 92–105% range) was obtained.

Gradient Retention Factor Concept Applied to Method Development for Peptide Analysis by Means of RP-HPLC.

Using the van Deemter model, the efficiency of three stationary phase systems in the analysis of a mixture of synthetic peptides was evaluated: (i) monolithic, (ii) packed, and (iii) core-shell columns, and it was shown that the efficiency of the monolithic column is superior to the others, specifically using it, the lowest values of H min (0.03 and 0.1 mm) were obtained, and additionally its efficiency was not significantly affected by increasing the flow. Using the concept of the gradient retention factor (k*), a method for chromatographic separation of a peptide complex mixture was designed, implemented, and optimized and then transferred from a packed column to a monolithic one. The results showed that it was possible to separate all components of the mixture using both evaluated columns; moreover, the analysis time was reduced from 70 to 10 min, conserving the critical pair resolution (1.4), by the transfer method using the k* concept. The method developed was tested against a mixture of doping peptides, showing that this method is efficient for separating peptides of various natures. This investigation is very useful for the development of methods for the analysis of complex peptide mixtures since it provides a systematic approach that can be extrapolated to different types of columns and instrumentation.

Separation of Molar Weight-Distributed Polyethylene Glycols by Reversed-Phase Chromatography—Analysis and Modeling Based on Isocratic Analytical-Scale Investigations

The separation of polyethylene glycols (PEGs) into single homologs by reversed-phase chromatography is investigated experimentally and theoretically. The used core–shell column is shown to achieve the baseline separation of PEG homologs up to molar weights of at least 5000 g/mol. A detailed study is performed elucidating the role of the operating conditions, including the temperature, eluent composition, and degree of polymerization of the polymer. Applying Martin’s rule yields a simple model for retention times that holds for a wide range of conditions. In combination with relations for column efficiency, the role of the operating conditions is discussed, and separations are predicted for analytical-scale chromatography. Finally, the approach is included in an efficient process model based on discrete convolution, which is demonstrated to predict with high accuracy also advanced operating modes with arbitrary injection profiles.

Characterization of complex photosynthetic pigment profiles in European deciduous tree leaves by sequential extraction and reversed-phase high-performance liquid chromatography.

Leaf pigments, including chlorophylls and carotenoids, are important biochemical indicators of plant photosynthesis and photoprotection. In this study, we developed, optimized, and validated a sequential extraction and liquid chromatography-diode array detection method allowing for the simultaneous quantification of the main photosynthetic pigments, including chlorophyll a, chlorophyll b, β-carotene, lutein, neoxanthin, and the xanthophyll cycle (VAZ), as well as the characterization of plant pigment derivatives. Chromatographic separation was accomplished with the newest generation of core–shell columns revealing numerous pigment derivatives. The sequential extraction allowed for a better recovery of the main pigments (+25 % chlorophyll a, +30 % chlorophyll b, +42 % β-carotene, and 61% xanthophylls), and the characterization of ca. 5.3 times more pigment derivatives (i.e., up to 62 chlorophyll and carotenoid derivatives including isomers) than with a single-step extraction. A broad working range of concentrations (300–2,000 ng.mL−1) was achieved for most pigments and their derivatives and the limit of detection was as low as a few nanograms per milliliter. The method also showed adequate trueness (RSD < 1%) and intermediate precision (RSD < 5%). The method was developed and validated with spinach leaves and their extracts. The method was successfully performed on leaf pigment extracts of European deciduous tree species. Within a case study using Fagus sylvatica L. leaves, pigment derivatives revealed a high within-individual tree variability throughout the growing season that could not be detected using the main photosynthetic pigments alone, eventually showing that the method allowed for the monitoring of pigment dynamics at unprecedented detail.

Rapid Determination of Preservatives in Cosmetics Using a Core-Shell Column.

Preservatives are frequently added to cosmetics to maintain product quality. Our laboratory quantifies 11 preservatives in cosmetics each year for regulatory purposes. In laboratories, many manufactures also analyze the preservatives in their products for quality control. To analyze many cosmetic samples, a rapid analysis method is required for efficiency. In this study, we developed a rapid method for the simultaneous determination of 11 regulated preservatives in cosmetics using a core-shell column by high-performance liquid chromatography. In this method, the 11 preservatives were separated within 17min, which was approximately half the time reported in the previous study. The peak resolution for each preservative was >2.6, the correlation coefficients of the calibration curves were >0.9988, the percent recoveries were 92.0-111.9% and the relative standard deviations were <3.5% (n = 3). The relative standard deviations among 6 researchers were <4.7%. Thus, it is an effective rapid determination method for the analysis of preservatives in cosmetics.

Boron nitride nanotubes for extraction of angiotensin receptor blockers from human plasma

The limitations of silica-based sorbents boosted the development of new extraction phases. In this study, boron nitride nanotubes functionalized with octadecyl groups were used for the first time as sorbent for extraction of losartan and valsartan, the most used angiotensin receptor blockers in the clinical practice, from human plasma. The nanotubes were synthesized using the chemical vapor deposition technique, purified by acid treatment, functionalized with octadecylamine in a microwave reactor, and characterized by different techniques. The functionalized nanotubes were packed in solid phase extraction cartridges. Extraction conditions were optimized by means of a 23 factorial design with center points. The separation was performed on a biphenyl core-shell (100 × 4.6 mm; 2.6 µm) column, using 0.1 % (v/v) triethylamine solution and methanol (pH 3.2) as mobile phase, at 0.7 mL/min, in gradient elution. The injection volume was 10 µL and fluorescence detection was performed at excitation and emission wavelengths of 250 and 375 nm, respectively. The developed method was validated according to Brazilian Health Regulatory Agency (ANVISA), United States Food and Drug Administration (US FDA) and European Medicines Agency (EMA) guidelines and presented selectivity, precision, accuracy, and linearity in the concentration ranges of 50–1200 ng/mL for losartan and 20–1700 ng/mL for valsartan. Recoveries higher than 80 % were obtained. The method was fit for the quantification of losartan in plasma samples from patients under antihypertensive therapy, being useful in therapeutic drug monitoring, pharmacokinetics and bioequivalence studies.

A simple UPLC-MS/MS assay with a core-shell column for the determination of exemestane in human plasma for clinical application.

Exemestane is one of the aromatase inhibitors and has been used to treat breast cancer by lowering estrogen levels. Accurate quantification of exemestane is important to set an optimal dose, and thus a simple assay for exemestane is developed by ultra-performance liquid chromatography with tandem mass spectrometer. Exemestane was extracted from human plasma samples (100 μL) by simple protein precipitation with acetonitrile/methanol (1/1, v/v). Interference peaks observed close to the elution of exemestane led us to use a core shell column for higher selectivity instead of totally porous columns. The extracts were chromatographed on CORTECS UPLC C18, under a gradient elution at a flow rate of 0.25 mL/min and detected in the selected reaction monitoring. Validation parameters were assessed in accordance with the bioanalytical guidelines using quality control samples. Exemestane in human plasma was quantifiable from 0.5 to 50 ng/mL with high extraction recovery and minimal matrix effects. Hemolyzed or hyperlipemic plasma did not impact the exemestane assay. Exemestane was stable in human plasma for 392 days at -15°C or below. The developed assay was robust and successfully applied to quantifying exemestane concentrations in human plasma to support a clinical trial.

Analytical scale supercritical fluid chromatography for the analysis of nine tocochromanols in 24 different cold-pressed plant oils: Method development, validation, and isolation of tocotrienols and plastochromanol-8

In the present study, four tocotrienol (T3) homologues and plastochromanol-8 (PC-8) were isolated from plant oils; Linum usitatissimum (PC-8), Bixa orellana (γ-T3 and δ-T3), and Triticum spelta bran (α-T3 and β-T3) by analytical scale supercritical fluid chromatography (SFC). The optimized method of tocopherols, tocotrienols, and PC-8 separation via SFC with UV detection was validated on a biphenyl core-shell column. The sample preparation conditions of the plant oils involved a simple dilution in 2-propanol (1:9, v/v), followed by direct injection into the SFC. For the analysis of 24 different plant oils we recommended the following final analytical scale SFC method conditions: 5 µL for the injection volume and sample oil:2-propanol dilution ratio of 1:9. The final assay developed facilitated the rapid (<15 min) and sensitive (limit of detection within the range of 2.5–7.4 μg/mL and the limit of quantitation within the range of 7.7–22.6 μg/mL) analyses of tocochromanols in obtained cold-pressed oils from twenty-four different plant species. Furthermore, the method was repeatable and reproducible with % RSD values in the context of standard retention times which ranged within 0.10–0.31 intraday and 0.59–0.79 interday.

Two Green Micellar HPLC and Mathematically Assisted UV Spectroscopic Methods for the Simultaneous Determination of Molnupiravir and Favipiravir as a Novel Combined COVID-19 Antiviral Regimen.

Following the spread of the COVID-19 pandemic crisis, a race was initiated to find a successful regimen for postinfections. Among those trials, a recent study declared the efficacy of an antiviral combination of favipiravir (FAV) and molnupiravir (MLP). The combined regimen helped in a successful 60% eradication of the SARS-CoV-2 virus from the lungs of studied hamster models. Moreover, it prevented viral transmission to cohosted sentinels. Because both medications are orally bioavailable, the coformulation of FAV and MLP can be predicted. The developed study is aimed at developing new green and simple methods for the simultaneous determination of FAV and MLP and then at their application in the study of their dissolution behavior if coformulated together. A green micellar HPLC method was validated using an RP-C18 core-shell column (5 μm, 150 × 4.6 mm) and an isocratic mixed micellar mobile phase composed of 0.1 M SDS, 0.01 M Brij-35, and 0.02 M monobasic potassium phosphate mixture and adjusted to pH 3.1 at 1.0 mL min−1 flow rate. The analytes were detected at 230 nm. The run time was less than five minutes under the optimized chromatographic conditions. Four other multivariate chemometric model methods were developed and validated, namely, classical least square (CLS), principal component regression (PCR), partial least squares (PLS-1), and genetic algorithm–partial least squares (GA–PLS-1). The developed models succeeded in resolving the great similarity and overlapping in the FAV and MLP UV spectra unlike the traditional univariate methods. All methods were organic solvent-free, did not require extraction or derivatization steps, and were applied for the construction of the simultaneous dissolution profile for FAV tablets and MLP capsules. The methods revealed that the amount of the simultaneously released cited drugs increases up until reaching a plateau after 15 and 20 min for FAV and MLP, respectively. The greenness was assessed on GAPI and found to be in harmony with green analytical chemistry concepts.

Development and validation of a direct HPLC method for the determination of salivary glutathione disulphide using a core shell column and post column derivatization with o-phthalaldehyde

Glutathione disulfide (GSSG) has been monitored in human saliva samples by an optimized and validated method that is based on liquid chromatography coupled to on-line post column derivatization. The analyte was separated from the sample matrix using a 100% aqueous mobile phase through a core–shell reversed phase column. Following optimization of the reaction using Box- Behnken experimental design and validation, GSSG was quantified accurately and selectively in the range of 100–2000 nmol L−1 with a LOD of 20 nmol L−1. GSSG was quantified in 15 out of 20 human saliva samples (75%) with a mean value of 860 nmol L−1 (150–4600 nmol L−1). Blocking of reduced Glutathione with N-ethylmaleimide ensured stability of the samples for at least 72 h at all temperatures examined.

Validation of a High-Performance Liquid Chromatography with Photodiode Array Detection Method for the Separation and Quantification of Antioxidant and Skin Anti-Aging Flavonoids from Nelumbo nucifera Gaertn. Stamen Extract.

Nelumbo nucifera Gaertn., or the so-called sacred lotus, is a useful aquatic plant in the Nelumbonaceae family that has long been used to prepare teas, traditional medicines as well as foods. Many studies reported on the phytochemicals and biological activities of its leaves and seeds. However, to date, only few studies were conducted on its stamen, which is the most important ingredient for herbal medicines, teas and other phytopharmaceutical products. Thus, this present study focuses on the following: (1) the application of high-performance liquid chromatography with photodiode array detection for a validated separation and quantification of flavonoids from stamen; (2) the Nelumbo nucifera stamen’s in vitro and in cellulo antioxidant activities; as well as (3) its potential regarding the inhibition of skin aging enzymes for cosmetic applications. The optimal separation of the main flavonoids from the stamen ethanolic extract was effectively achieved using a core-shell column. The results indicated that stamen ethanolic extract has higher concentration of in vitro and in cellulo antioxidant flavonoids than other floral components. Stamen ethanolic extract showed the highest protective effect against reactive oxygen/nitrogen species formation, as confirmed by cellular antioxidant assay using a yeast model. The evaluation of potential skin anti-aging action showed that the stamen extract has higher potential to inhibit tyrosinase and collagenase compared with its whole flower. These current findings are the first report to suggest the possibility to employ N. nucifera stamen ethanolic extract as a tyrosinase and collagenase inhibitor in cosmetic applications, as well as the utility of the current separation method.

Determination of the Chemical Stability of Dapagliflozin by LC/DAD and MS/MS Methods.

A simple and fast stability-indicating liquid chromatographic method with diode array detection (DAD) was developed and validated for the determination of dapagliflozin (DAPA) in bulk and tablets, in the presence of its major degradation products (DP). The drug was subjected to hydrolytic, oxidative, photolytic, thermal and humidity/thermal stress conditions, showing significant degradation under humidity/thermal with the formation of two DP, which were preliminarily identified by liquid chromatography with diode array detector coupled with electrospray ionization-tandem mass spectrometry (HPLC-DAD-ESI-MS/MS). Chromatographic separation of dapagliflozin and its DP was achieved with a core-shell RP-18 column, using acetonitrile and water as mobile phase in isocratic elution mode. The described method was linear over a range of 50-150μg/mL. For precision, the relative standard deviation (RSD) was <1.3%, the recovery was 99.64-100.11%, and the assay demonstrated adequate selectivity. The degradation kinetics of dapagliflozin was evaluated corresponding to first-order under thermal and humidity/thermal stress conditions. Dapagliflozin was well resolved from its drug products showing the power of stability-indicating of the method. The results showed that the proposed method was found to be suitable for routine analysis, quantitative determination and the stability study of dapagliflozin in pharmaceutical samples.

Mass spectrometry-based targeted proteomics method for the quantification of clinically relevant drug metabolizing enzymes in human specimens

Biotransformation by phase I and II metabolizing enzymes represents the major determinant for the oral bioavailability of many drugs. To estimate the pharmacokinetics, data on protein abundance of hepatic and extrahepatic tissues, such as the small intestine, are required. Targeted proteomics assays are nowadays state-of-the-art for absolute protein quantification and several methods for quantification of drug metabolizing enzymes have been published. However, some enzymes remain still uncovered by the analytical spectra of those methods. Therefore, we developed and validated a quantification assay for two carboxylesterases (CES-1, CES-2), 17 cytochrome P450 enzymes (CYP) (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4, CYP3A5, CYP3A7, CYP4F2, CYP4F12, CYP4A11) and five UDP-glucuronosyltransferases (UGTs) (UGT1A1, UGT1A3, UGT2B7, UGT2B15, UGT2B17). Protein quantification was performed by analyzing proteospecific surrogate peptides after tryptic digestion with stable isotope-labelled standards. Chromatographic separation was performed on a Kinetex® 2.6 µm C18 100 Å core–shell column (100 × 2.1 mm) with a gradient elution using 0.1% formic acid and acetonitrile containing 0.1% formic acid with a flow rate of 200 µl/min. Three mass transitions were simultaneously monitored with a scheduled multiple reaction monitoring (sMRM) method for each analyte and standard. The method was partly validated according to current bioanalytical guidelines and met the criteria regarding linearity (0.1–25 nmol/L), within-day and between-day accuracy and precision as well as multiple stability criteria. Finally, the developed method was successfully applied to determine the abundance of the aforementioned enzymes in human intestinal und liver microsomes. Our work offers a new fit for purpose method for the absolute quantification of CES, CYPs and UGTs in various human tissues and can be used for the acquisition of data for physiologically based pharmacokinetic modelling.

Analysis of chemical components of Chinese medicine Ligustici Radix by achiral-chiral liquid chromatography-predictive multiple reaction monitoring

中药毛前胡为伞形科植物短片藁本Ligusticum brachylobum Franch.的干燥根,主要用于治疗风热咳嗽痰多、痰热喘满、咯痰黄稠等证,富含香豆素类化学成分,含有多组对映异构体和非对映异构体。为了深入研究毛前胡的化学成分组成,特别是对映异构体的组成,研究建立了非手性-手性色谱-预测多反应监测法(achiral-chiral-LC predictive MRM),同步实现毛前胡化学成分的化学选择性和立体选择性分离,以及高灵敏度定性分析。非手性色谱和手性液相色谱-串联质谱系统结合了RP-C18色谱柱的高效化学选择性分离能力以及手性色谱柱的立体选择性优势,有效避免了中心切割非手性-手性二维液相色谱构造复杂、重现性难以满足定量要求等缺陷。采用小内径核-壳型RP-C18色谱柱作为前端化学分离柱,实现结构类似香豆素的高效化学选择性分离;采用反相大内径AD-RH手性色谱柱,实现对映异构体的手性拆分;采用预测多反应监测模式,实现化学成分的高灵敏度检出;利用增强子离子扫描模式(EPI)采集各色谱峰的二级质谱信息,鉴定化学结构。通过定量离子对、定性离子对及两者的比值,判定是否为对映异构体。利用所构建的非手性-手性色谱耦联系统从毛前胡中共鉴定出60个化学成分,其中8对香豆素对映异构体得到了良好分离。本研究为毛前胡以及含有对映异构体中药的深入定性、定量分析提供可靠的方法。

Simplification of Moment Analysis Procedure for Kinetic Study of Chromatographic Behavior of Core-shell Particles.

The moment analysis method for chromatographic behavior in core-shell columns was simplified. Mass-transfer phenomena other than intra-stationary phase diffusion are analyzed while considering that the packing materials are spherical particles. The manner of intra-stationary phase diffusion is analyzed while assuming a hypothetical flat plate. For most core-shell particles commercially available, the geometry of a spherical thin layer can be supposed as a hypothetical flat plate with a relative error of less than ca. 2% because the thickness of the shell layer is sufficiently smaller than the diameter of whole particle. This supposition makes moment analysis easier because the moment equations for flat plates are simpler than those strictly developed for core-shell particles. Some chromatographic data measured using a core-shell column were analyzed by the simple moment analysis method to confirm its usefulness. It was demonstrated that the method is effective for a preliminary study of mass-transfer kinetics in core-shell columns.

Determination of Nateglinide in Tablet Formulation by HPLC Using a Pentafluorophenyl Core-Shell Column

Nateglinide is an insulin secretagogue which has been used for the treatment of Type 2 Diabetes mellitus. A simple analytical methodology for determination of nateglinide in tablet formulation is described. An isocratic reversed phase high performance liquid chromatographic (HPLC) method was developed using Kinetex pentafluorophenyl (PFP) (5μm particle size and 4.6 x 150 mm id) core-shell column as stationary phase and 0.05 M Na2HPO4 (pH=2.0) / methanol (30:70 v/v) mixture as mobile phase with the flow rate of 1.0 mL min-1 and diode-array detector at 215 nm. Column was termostated at 22°C. Under these conditions nateglinide was eluted with a symmetrical peak shape and the retention time was 4.779 minutes. The system suitability parameters such as tailing factor, capacity factor and theoretical plate were evaluted. The method was validated as per International Council on Harmonisation (ICH) guidelines. Linearity was obtained in the 2.54 - 40.70 μg mL-1 concentration range with equation y= 10.818x + 4.7048 (R2= 0.998). Limit of detection and limit of quantification were 1.07 and 2.54 μg mL-1. Accuracy of the method was tested by recovery studies which were in the range of 98.78 - 100.76 %. The intra-day and inter-day precision studies were carried out and the relative standard deviation values of peak areas were within ICH limits. The proposed method was successfully applied to the determination of nateglinide in tablet dosage form.