Analysis of pharmaceutical impurities using multi‐heartcutting 2D LC coupled with UV‐charged aerosol MS detection

To overcome challenges in HPLC impurity analysis of pharmaceuticals, we developed an automated online multi‐heartcutting 2D HPLC system with hyphenated UV‐charged aerosol MS detection. The first dimension has a primary column and the second dimension has six orthogonal columns to enhance flexibility and selectivity. The two dimensions were interfaced by a pair of switching valves equipped with six trapping loops that allow multi‐heartcutting of peaks of interest in the first dimension and also allow “peak parking.” The hyphenated UV‐charged aerosol MS detection provides comprehensive detection for compounds with and without UV chromophores, organics, and inorganics. It also provides structural information for impurity identification. A hidden degradation product that co‐eluted with the drug main peak was revealed by RP × RP separation and thus enabled the stability‐indicating method development. A poorly retained polar component with no UV chromophores was analyzed by RP × hydrophilic interaction liquid chromatography separation with charged aerosol detection. Furthermore, using this system, the structures of low‐level impurities separated by a method using nonvolatile phosphate buffer were identified and tracked by MS in the second dimension.     

Improved sulfoalkylbetaine‐based organic‐silica hybrid monolith for high efficient hydrophilic interaction liquid chromatography of polar compounds

A novel sulfoalkylbetaine‐based zwitterionic organic‐silica hybrid monolith was synthesized by using 3‐dimethyl‐(3‐(N‐methacrylamido) propyl) ammonium propane sulfonate (DMMPPS, neutral sulfoalkyl‐betaine monomer). The added amount of zwitterionic monomer was significantly increased when DMMPPS was used instead of the conventionally used acidic sulfoalkyl‐betaine monomer, that is, the N,N‐dimethyl‐N‐ methacryloxyethyl‐N‐(3‐sulfopropyl) ammonium betaine, and this led to a significantly improved hydrophilicity of the monolith. The DMMPPS‐based organic‐silica hybrid monolith exhibited good mechanical stability and excellent separation performance. About ～20 μm plate height (corresponding to column efficiency of ～50 000 plates/m) was obtained for nucleoside at the linear velocity of 1 mm/s. The proposed monolithic column was successfully applied to separate purines/pyrimidines, nucleotides, and tryptic digest of bovine hemoglobin in a nano‐HILIC mode, and the results demonstrated that such monolith has the potential for separation of a variety of hydrophilic substances.     

HILIC LC‐MS for the determination of 2′‐C‐methyl‐cytidine‐triphosphate in rat liver

A very accurate and selective LC‐MS/MS method was developed and validated for the quantification of 2′‐C‐modified nucleoside triphosphate in liver tissue samples. An efficient pretreatment procedure of liver tissue samples was developed, using a fully automated SPE procedure with 96‐well SPE plate (weak anion exchange sorbent, 30 mg). Nucleotide hydrophilic interaction chromatography has been performed on an aminopropyl column (100 mm×2.0 mm, 3 μm) using a gradient mixture of ACN and ACN/water (5:95 v/v) with 20 mM ammonium acetate at pH 9.45 as mobile phase at 300 μL/min flow rate. The 2′‐C‐modified nucleoside triphosphate was detected in the negative ESI mode in multiple reaction monitoring (MRM) mode. Calibration curve was linear over the 0.05–50 μM concentration range. Satisfying results, confirming the high reliability of the established LC‐MS/MS method, were obtained for intraday precision (CV = 2.5–9.1%) and accuracy (92.6–94.8%) and interday precision (CV = 9.6–11.5%) and accuracy (94.4–102.4%) as well as for recovery (82.0–112.6%) and selectivity. The method has been successfully applied for pharmacokinetic studies of 2′‐C‐methyl‐cytidine‐triphosphate in liver tissue samples.     

Optimization of o‐phtaldialdehyde/2‐mercaptoethanol postcolumn reaction for the hydrophilic interaction liquid chromatography determination of memantine utilizing a silica hydride stationary phase

A rapid procedure for the determination of memantine based on hydrophilic interaction chromatography with fluorescence detection was developed. Fluorescence detection after postcolumn derivatization with o‐phtaldialdehyde/2‐mercaptoethanol was performed at excitation and emission wavelengths of 345 and 450 nm, respectively. The postcolumn reaction conditions such as reaction temperature, derivatization reagent flow rate, and reagents concentration were studied due to steric hindrance of amino group of memantine. The derivatization reaction was applied for the hydrophilic interaction liquid chromatography method which was based on Cogent Silica‐C stationary phase with a mobile phase consisting of a mixture of 10 mmol/L citric acid and 10 mmol/L o‐phosphoric acid (pH 6.0) with acetonitrile using an isocratic composition of 2:8 v/v. The benefit of the reported approach consists in a simple sample pretreatment and a quick and sensitive hydrophilic interaction chromatography method. The developed method was validated in terms of linearity, accuracy, precision, and selectivity according to the International Conference on Harmonisation guidelines. The developed method was successfully applied for the analysis of commercial memantine tablets.     

Comprehensive analysis of pharmaceutical products using simultaneous mixed‐mode (ion‐exchange/reversed‐phase) and hydrophilic interaction liquid chromatography

Liquid chromatographic assays were developed using a mixed‐mode column coupled in sequence with a hydrophilic interaction liquid chromatography column to allow the simultaneous comprehensive analysis of inorganic/organic anions and cations, active pharmaceutical ingredients, and excipients (carbohydrates). The approach utilized dual sample injection and valve‐mediated column switching and was based upon a single high‐performance liquid chromatography gradient pump. The separation consisted of three distinct sequential separation mechanisms, namely, (i) ion‐exchange, (ii) mixed‐mode interactions under an applied dual gradient (reversed‐phase/ion‐exchange), and (iii) hydrophilic interaction chromatography. Upon first injection, the Scherzo SS C₁₈ column (Imtakt) provided resolution of inorganic anions and cations under isocratic conditions, followed by a dual organic/salt gradient to elute active pharmaceutical ingredients and their respective organic counterions and potential degradants. At the top of the mixed‐mode gradient (high acetonitrile content), the mobile phase flow was switched to a preconditioned hydrophilic interaction liquid chromatography column, and the standard/sample was reinjected for the separation of hydrophilic carbohydrates, some of which are commonly known excipients in drug formulations. The approach afforded reproducible separation and resolution of up to 23 chemically diverse solutes in a single run. The method was applied to investigate the composition of commercial cough syrups (Robitussin®), allowing resolution and determination of inorganic ions, active pharmaceutical ingredients, excipients, and numerous well‐resolved unknown peaks.     

Serial coupling of reversed‐phase and zwitterionic hydrophilic interaction LC/MS for the analysis of polar and nonpolar phenols in wine

In the present study, an easy and efficient method based on the serial coupling of analytical reversed‐phase and zwitterionic hydrophilic interaction liquid chromatography was developed for the simultaneous separation of polar and nonpolar phenols occurring in wine. The zwitterionic hydrophilic column was connected in series to the reversed‐phase one via a T‐piece, with which the ACN content in eluent of the second dimension was increased, in order to cope the solvent strength incompatibility between the two columns. The final mobile phase at low‐flow rate (≤0.5 mL/min), high‐ACN content (90%), and low‐salt concentration was directed to an ESI‐TOF‐MS , for high accurate mass detections. The developed method was applied for the identification of target phenols in several wines. Retention time and peak width intra‐ and interday repeatability studies proved the reliability of the method for the simultaneous analysis of all the polar and nonpolar analytes in wine. The serial reversed‐phase/zwitterionic hydrophilic interaction liquid chromatography coupling offered the possibility to enlarge the number of identified compounds and it represents a valid approach for nontarget analysis of complex samples by a single injection.     

Rapid analysis of constituents of Radix Cyathulae using hydrophilic interaction‐reverse phase LC‐MS

A hydrophilic interaction chromatography (HILIC) and reverse‐phase liquid chromatography (RPLC) coupled with electrospray TOF MS method was developed for the analysis and characterization of constituents in the radix of Cyathula officinalis Kuan. Separation parameters of HILIC such as buffer pH, mobile phase strength, and organic modifier were evaluated. Fructose, glucose, and sucrose were identified by HILIC‐ESI/TOF MS. Reverse‐phase liquid chromatography‐ESI/TOF MS were applied for quick and sensitive identification of major saponins in Cyathula officinalis. In‐source collision‐induced dissociation has been performed to elucidate the fragmentation pathways of oleanane‐, hederagenin‐, and gypsogmin‐type saponins. Twelve saponins were characterized in this plant for the first time, and four of them were presumed to be new compounds. In addition, one phytoecdysteroid (cyasterone) and one coumarin (6,7‐dimethoxycoumarin) were detected at the same time. The present method was capable of rapid characterizing and providing structure information of constituents from herbal drugs.     

Pharmacokinetics of methyl salicylate‐2‐O‐β‐D‐lactoside,a novel salicylic acid analog isolated from Gaultheria yunnanensis,in dogs

Methyl salicylate‐2‐O‐β‐D‐lactoside (MSL), a natural salicylate derivative of Gaultheria yunnanensis (Franch.) Rehder (G. yunnanensis), has been shown to provide a beneficial anti‐inflammatory effect in animal models. Studies on the pharmacokinetics and bioavailability of MSL can provide both a substantial foundation for understanding its mechanism and empirical evidence to support its use in clinical practice. A simple and sensitive high‐performance liquid chromatography (HPLC) method, coupled with ultraviolet analyte detection, was developed for determining the concentration of MSL and its metabolite in beagle plasma. Chromatographic separation was achieved on a Agilent Zorbax SB‐C₁₈ column (5 μm ,4.6 × 250 mm). The mobile phase consisted of aqueous solution containing 0.1% phosphoric acid and acetonitrile (82:90, v/v), at a flow rate of 1 mL/min. Validation of the assay demonstrated that the developed HPLC method was sensitive, accurate and selective for the determination of MSL and its metabolite in dog plasma. After orally administering three doses of MSL, it could no longer be detected in dog plasma and its metabolite, salicylic acid, was detected. Salicylic acid showed a single peak in the plasma concentration–time curves and linear pharmacokinetics following the three oral doses (r² > 0.99). In contrast, only MSL was detected in plasma following intravenous administration. These results will aid in understanding the pharmacological significance of MSL. The developed method was successfully used for evaluation of the oral and intravenous pharmacokinetic profile of MSL in dogs. Copyright © 2013 John Wiley & Sons, Ltd.     

Comparison of reversed‐phase liquid chromatography and hydrophilic interaction chromatography for the fingerprint analysis of Radix isatidis

Radix isatidis is a famous anti‐influenza virus herbal medicine traditionally taken as a water decoction. However, the chemical fingerprint analysis of Radix isatidis is dominantly based on RPLC, from which it is difficult to obtain fingerprint information of hydrophilic compounds. Here, we developed the separation of Radix isatidis by RPLC and hydrophilic interaction chromatography, comparing the traditional RPLC fingerprint with the hydrophilic interaction chromatography fingerprint. Besides, an anti‐viral assay of Radix isatidis was conducted to evaluate its efficacy. The fingerprint–efficacy relationships between the fingerprints and the anti‐viral activity were further investigated with principal component regression analysis. The results showed that the anti‐viral activity correlated better with the hydrophilic interaction chromatography fingerprint than with the RPLC fingerprint. This study indicates that hydrophilic interaction chromatography could not only be a complementary method to increase the fingerprint coverage of conventional RPLC fingerprint, but also can better represent the efficacy and quality of Radix isatidis.     

A facile and efficient one‐step strategy for the preparation of β‐cyclodextrin monoliths

A novel, facile, and efficient one‐step copolymerization strategy was developed for the preparation of β‐cyclodextrin (β‐CD) methacrylate monolithic columns using click chemistry. The novel mono‐(1H‐1,2,3‐triazol‐4‐ylmethyl)‐2‐methylacryl‐β‐CD monomer was synthesized by a click reaction between propargyl methacrylate and mono‐6‐azido‐β‐CD, and then monolithic columns were prepared through a one‐step in situ copolymerization of the mono‐(1H‐1,2,3‐triazol‐4‐ylmethyl)‐2‐methylacryl‐β‐CD monomer and ethylene dimethacrylate. The physicochemical properties and column performance of the fabricated monolithic columns were characterized by elemental analysis, SEM, and micro‐HPLC. Satisfactory column permeability, efficiency, and separation performance were obtained for the optimized poly(mono‐(1H‐1,2,3‐triazol‐4‐ylmethyl)‐2‐methylacryl‐β‐CD‐co‐ethylene dimethacrylate) monolithic columns. Additionally, typical hydrophilic interaction chromatography retention behavior was observed on the monoliths at high acetonitrile content in the mobile phase. Although the enantioselectivity of our monolithic columns did not meet the level of other reported β‐CD monolithic columns, this one‐step strategy based on click chemistry still provides an interesting and effective model as it offers the possibility to easily prepare related novel CD methacrylate monoliths through a one‐step copolymerization strategy.     

2－脱氧－烟酰胺基－β－D－氨基葡萄糖的合成和表征

A new nicotinic acid derivative,2-deoxy-2-nicotinoylamido-β-D-glucopyranose, was synthesized with β-configuration exclusively. The structure and properties of the product were characterized by ^1H NMR, PT-IR, MS, DSC and polarimeter. The details of ^1H NMR spectrum and the mass spectrum proved that there are a great amount of hydrogen bonds in the product.     

A Novel Synthesis of 2-Amino-2-deoxy-D-gluconic Acid and Its Complexation with Cu（Ⅱ） in Alkaline Medium

A novel synthesis of the functional carbohydrate 2-amino-2-deoxy-D-gluconic acid was introduced and itscomplex formation with Cu(Ⅱ)was investigated to obtain the stability constant for its further applications to thefood and pharmaceutical industries.The equilibrium was investigated by spectrophotometric measurements andprocessed by dual-series linear regression method.Results:the yield of 2-amino-2-deoxy-D-gluconic acid is 70%.The complexation molar ratio is 1:2,the molar apsorptivity of the complex is 39.906 L·mol~(-1)·cm~(-1) at 630 nm,and the stability constant β_n is 6.24×10~5.     

Simultaneous determination of 20 components in red wine by LC‐MS: Application to variations of red wine components in decanting

The decanting of red wines has a long tradition in red wine service from the perspective of modifying the aroma or taste of a wine. A simple and sensitive liquid chromatography‐mass spectrometry method was developed for the simultaneous determination of 20 organic acids and polyphenols in decanting red wine. The separation was performed on a Diamonsil C₁₈ column (250 mm × 4.6 mm, 5 μm) using a mobile phase composed of methanol‐0.1% acetic acid under gradient elution. Analysis was performed in selected ion monitoring mode with negative electrospray ionization interface. All the linear regressions showed good linear relationships (r² > 0.9973) between the peak area and concentration of each marker. The assay was reproducible with overall intra and interday variation of less than 5.0%. The recoveries for the quantified compounds were observed over the range of 92.1–108.3% with RSD values less than 5.7%. The method developed was successfully applied to determine the variations of the 20 components in red wine after decanting in different conditions. Concentrations of most organic acids and polyphenols investigated in the red wine were decreased in decanting. In addition, increment of duration, temperature, and light intensity would intensify the changes.     

Determination of paroxetine in serum treated with simple pretreatment by pre‐column high‐performance liquid chromatography using 4‐(5,6‐dimethoxy‐2‐phthalimidinyl)‐2‐methoxyphenylsulfonyl chloride as a fluorescent labeling reagent

The therapeutic drug monitoring of paroxetine could be used to optimize the pharmacological treatment of depressed patients. A simple and sensitive high‐performance liquid chromatography procedure was developed for the determination of paroxetine in serum. After simple pretreatment of serum (50 μL) with acetonitrile and o‐phthalaldehyde, paroxetine was derivatized with 4‐(5,6‐dimethoxy‐2‐phthalimidinyl)‐2‐methoxyphenylsulfonyl chloride at 70°C for 20 min in borate buffer (0.1 mol/L, pH 8.0) to produce a fluorescent product. The derivative was separated on a reversed‐phase column at 40°C for stepwise elution with (A) acetic acid (10 mmol/L) and (B) acetonitrile. The flow rate was 1.0 mL/min. The fluorescence intensity was monitored at excitation and emission wavelengths of 320 and 400 nm, respectively. The within‐day and day‐to‐day relative standard deviations were 3.0–3.4 and 2.7–8.3%, respectively. The detection limit of paroxetine was 8.3 fmol at a signal‐to‐noise ratio of 3. As the proposed method that only requires a small quantity of serum (50 μL) is simple, sensitive and reproducible, it would be useful for clinical and biochemical research as well as drug monitoring. Copyright © 2013 John Wiley & Sons, Ltd.     

On‐line 2D‐LC‐ESI/MS/MS determination of rifaximin in rat serum

A highly sensitive and selective on‐line two‐dimensional reversed‐phase liquid chromatography/electrospray ionization–tandem mass spectrometry (2D‐LC‐ESI/MS/MS) method was developed and validated to determine rifaximin in rat serum by direct injection. The 2D‐LC‐ESI/MS/MS system consisted of a restricted access media column for trapping proteins as the first dimension and a Waters C₁₈ column as second dimension using 0.1% aqueous acetic acid:acetonitrile as mobile phase in a gradient elution mode. Rifampacin was used as an internal standard. The linear dynamic range was 0.5–10 ng/mL (r² > 0.998). Acceptable precision and accuracy were obtained over the calibration range. The assay was successfully used in analysis of rat serum to support pharmacokinetic studies. Copyright © 2009 John Wiley & Sons, Ltd.     

Enantiomeric separation of 2‐arylpropionic acid nonsteroidal anti‐inflammatory drugs by HPLC with hydroxypropyl‐β‐cyclodextrin as chiral mobile phase additive

The enantio‐separations of eight 2‐arylpropionic acid nonsteroidal anti‐inflammatory drugs (2‐APA NSAIDs) were established using reversed‐phase high‐performance liquid chromatography with hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) as chiral mobile phase additive for studying the stereoselective skin permeation of suprofen, ketoprofen, naproxen, indoprofen, fenoprofen, furbiprofen, ibuprofen and carprofen. The effects of the mobile phase composition, concentration of HP‐β‐CD and column temperature on retention and enantioselective separation were investigated. With 2‐APA NSAIDs as acidic analytes, the retention times and resolutions of the enantiomers were strongly related to the pH of the mobile phase. In addition, both the concentration of HP‐β‐CD and temperature had a great effect on retention time, but only a slight or almost no effect on resolutions of the analytes. Enantioseparations were achieved on a Shimpack CLC‐ODS (150 × 4.6 mm i.d., 5 μm) column. The mobile phase was a mixture of methanol and phosphate buffer (pH 4.0–5.5, 20 mM) containing 25 mM HP‐β‐CD. This method was flexible, simple and economically advantageous over the use of chiral stationary phase, and was successfully applied to the enantioselective determination of the racemic 2‐APA NSAIDs in an enantioselective skin permeation study. Copyright © 2009 John Wiley & Sons, Ltd.     

Enantioselective isolation of methyl jasmonate using permethyl‐β‐cyclodextrin HPLC

A method based on the use of HPLC for the enantioselective resolution of the four stereoisomers of methyl jasmonate (MJ) with no need for the previous formation of the diastereoisomers is developed. To that end, a Nucleodex‐β‐PM column as well as an optimization process considering different flow rates and mobile phase compositions were required. As a result, 0.8 mL/min and 55:45 methanol/water composition were the conditions selected to carry out the separation of the stereoisomers. Isolation of pure (–)‐ and (+)‐MJ was accomplished by collecting the HPLC fractions corresponding to their elution time. SPE was subsequently used to concentrate and change the solvent of the HPLC fractions collected. Chiral GC and polarimetry were additionally employed to evaluate the purity and optical rotation, respectively, of the enantiomers separated. The results found in this study are particularly relevant considering that MJ stereoisomers are not commercially available.     

MeCAT peptide labeling for the absolute quantification of proteins by 2D‐LC‐ICP‐MS

Metal‐Coded Affinity Tags (MeCAT) reagents were devised for the absolute quantification of labeled proteins and peptides using inductively coupled plasma mass spectrometry (ICP‐MS). After the recent publication of quantification approaches for digested proteins, this work presents a multidimensional strategy for the application of MeCAT to samples which require higher chromatographic resolution. Two‐dimensional separations based on strong cation exchange (SCX) and reversed‐phase (RP) chromatography, were used for the quantification of lysozyme, bovine serum albumin and transferrin after tryptic digestion. The elution protocols were optimized to improve the resolution of the MeCAT‐labeled peptides which led to faster elutions in SCX and longer retention times in RP compared with unlabeled peptides. The optimized method provided enough resolution for the samples analyzed. Peptides losses during the whole procedure were studied. Although recoveries of greater than 90% were found in the RP dimension, important global losses in the two‐dimensional offline approach forced us to use specific internal standards, in this case MeCAT‐labeled standard peptides. External calibration and label‐specific isotope dilution analysis (IDA) were tested and compared as possible quantification techniques. While both techniques showed accurate and precise determinations, the label‐specific IDA technique resulted in more straightforward measurements and more affordable external calibrations. Finally, simultaneous quantification of three different samples labeled with different lanthanides was successfully performed demonstrating the potential of MeCAT combined with ICP‐MS for multiplexing. Electrospray ionization mass spectrometry techniques provided the structural information needed for the identification of the labeled species. Copyright © 2012 John Wiley & Sons, Ltd.     

Preparation of a novel carboxyl stationary phase by “thiol‐ene” click chemistry for hydrophilic interaction chromatography

A novel carboxyl‐bonded silica stationary phase was prepared by “thiol‐ene” click chemistry. The resultant Thiol‐Click‐COOH phase was evaluated under hydrophilic interaction liquid chromatography (HILIC) mobile phase conditions. A comparison of the chromatographic performance of Thiol‐Click‐COOH and pure silica columns was performed according to the retention behaviors of analytes and the charged state of the stationary phases. The results indicated that the newly developed Thiol‐Click‐COOH column has a higher surface charge and stronger hydrophilicity than the pure silica column. Furthermore, the chromatographic behaviors of five nucleosides on the Thiol‐Click‐COOH phase were investigated in detail. Finally, a good separation of 13 nucleosides and bases, and four water‐soluble vitamins was achieved.     

Magnetic solid‐phase extraction of pyrethroid pesticides in environmental water samples with CoFe2O4‐embedded porous graphitic carbon nanocomposites

Magnetic CoFe₂O₄‐embedded porous graphitic carbon nanocomposites were prepared through a facile solid‐phase thermal reaction with NaCl as a template. The material was applied in the magnetic solid‐phase extraction process coupled with high performance liquid chromatography with a diode array detector to detect the trace fenpropathrin, cyhalothrin, S‐fenvalerate, and bifenthrin in different water samples. The synthesis conditions of nanomaterial including glucose concentration and calcination time on extraction performance for pyrethroid pesticides have been investigated. Different magnetic solid‐phase extraction parameters have been studied, such as the nanomaterial amount, solution pH, eluent types, adsorption time, and the reusability. Under the optimum conditions, good recoveries (80.2–110.9%) were achieved with relative standard deviations of 0.2–5.8%. There are probably hydrophobic interactions and dipole–dipole attractions between nanocomposites and the analytes.