Mobile Phase Parameters Research Articles

Determination of eight neonicotinoid pesticides in wastewater by solid phase extraction combined with liquid chromatography-tandem mass spectrometry

Neonicotinoid pesticides are a relatively new class of pesticides that have garnered significant attention owing to their potential ecological risks to nontarget organisms. A method combining solid phase extraction with liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) was developed for the rapid and accurate detection of eight neonicotinoid pesticides (dinotefuran, E-nitenpyram, thiamethoxam, clothianidin, imidacloprid, imidaclothiz, acetamiprid, and thiacloprid) in wastewater. The chromatographic mobile phase and MS parameters were selected, and a single-factor method was used to determine the optimal column type, extraction volume, sample loading speed, and pH for SPE. The optimal parameters were as follows: column type, HLB column (500 mg/6 mL); sample extraction volume, 500 mL; sample loading speed, 10 mL/min; and sample pH, 6-8. The matrix effects of the wastewater samples were reduced by optimizing the chromatographic gradient-elution program, examining the dilution factor of the samples, and using the isotope internal standard calibration method. Prior to analysis, the wastewater samples were diluted 5-fold with ultrapure water for pretreatment. Subsequently, 2 mmol/L ammonium acetate aqueous solution containing 0.1% (v/v) formic acid and methanol was used as mobile phases for gradient elution on a ZORBAX Eclipse Plus C18 column (100 mm×2.1 mm, 1.8 μm). The samples were quantified using positive-ion multiple reaction monitoring (MRM) mode for 10 min. Imidacloprid-d4 was used as the isotope internal standard. The SPE process was further optimized by applying response surface methodology to select the type and mass of rinsing and elution solvents. The optimal pretreatment of the SPE column included rinsing with 10% methanol aqueous solution and elution with methanol-acetonitrile (1∶1, v/v) mixture (7 mL). The eight neonicotinoid pesticides showed satisfactory linearity within the relevant range, with linear correlation coefficients (r) all greater than 0.9990. The method detection limits (MDLs) ranged from 0.2 to 1.2 ng/L, and the method quantification limits (MQLs) ranged from 0.8 to 4.8 ng/L. The average recoveries of the eight neonicotinoid pesticides were in the range of 82.6%-94.2% at three spiked levels, with relative standard deviations (RSDs) ranging from 3.9% to 9.4%. Finally, the optimized method was successfully applied to analyze wastewater samples collected from four sewage treatment plants. The results indicated that the eight neonicotinoid pesticides could be generally detected at concentrations ranging from not detected (ND) to 256 ng/L. The developed method has a low MDL and high accuracy, rendering it a suitable choice for the trace detection of the eight neonicotinoid pesticides in wastewater when compared with other similar methods. The proposed method can be utilized to monitor the environmental impact and assess the potential risks of neonicotinoid pesticides in wastewater, thus promoting the protection of nontarget organisms and the sustainable use of these pesticides in agriculture.

A versatile economic strategy by HPLC–CAD for quantification of structurally diverse markers in quality control of Shengmai Formula from raw materials to preparations

BackgroundShengmai Formula (SMF), a classic formula in treating Qi-Yin deficiency, is composed of Ginseng Radix et Rhizoma Rubra (GRR), Ophiopogon Radix (OR), and Schisandra chinensis Fructus (SC), and has been developed into various dosage forms including Shengmai Yin Oral Liquid (SMY), Shengmai Capsules (SMC), and Shengmai Injection (SMI). The pharmacological effects of compound Chinese medicine are attributed to the integration of multiple components. Yet the quality criteria of SMF are limited to monitoring schisandrol A or ginsenosides Rg1 and Re, but none for OR. Since the complexity of raw materials and preparations, establishing a economical and unified method for SMF is challenging. It is urgent to simultaneously quantify multiple components with different structures using a universal method for quality control of SMF. Charged aerosol detector (CAD) overcame the above shortcomings owing to its characteristics of high responsiveness, nondiscrimination, and low cost. PurposeWe aimed to establish a versatile analysis strategy using HPLC–CAD for simultaneously quantifying the structurally diverse markers in quality control of SMF from raw materials to preparations. MethodBy optimizing the column, mobile phase, column temperature, flow rate, and CAD parameters, a HPLC-CAD method that integrated multi-component characterization, authenticity identification, transfer information of raw materials and quantitative determination of Shengmai preparations was established. ResultsIn total 50 components from SMF were characterized (28 in GRR, 13 in SC, and 9 in OR). The differences in raw materials between species of SC and Schisandrae sphenantherae Fructus (SS), processing methods of Ginseng Radix (GR) and GRR, and locations of OR from Sichuan (ORS) and Zhejiang (ORZ) were compared. Fourteen components in 19 batches of SMY, SMC and SMI from different manufacturers were quantified, including 11 ginsenosides and 3 lignans. The multivariate statistical analysis results further suggested that Rb1, Rg1 and Ro were the main differences among Shengmai preparations. ConclusionThe established versatile analysis strategy based on HPLC–CAD was proven sensitive, simple, convenient, overcoming the discriminatory effect of UV detector, revealing the composition and transfer information of SMF and applicable for authentication of the ingredient herbs and improving the quality of Shengmai preparations.

Improved adeno-associated virus empty and full capsid separation using weak partitioning multi-column AEX chromatography.

Recombinant adeno-associated virus (rAAV) empty and full capsid separation has been a topic of interest in the rAAV gene therapy community for many years and the anion exchange chromatography (AEX) step has undergone various process optimizations to improve rAAV empty capsid separation, including AEX stationary phase, mobile phase, and process parameters. Here, we present a new AEX method that employs both weak partitioning chromatography (WPC) and multi-column chromatography (MCC) to achieve improved full rAAV percentage in the AEX pool. The WPC technology allows empty rAAV to be displaced by full rAAV during loading, while the MCC technology enables parallel column processing which further increases AEX step productivity. Our results show that, compared to baseline AEX batch chromatography, the AEX-WPC-MCC method demonstrated improvements in both AEX pool full rAAV percentage (∼ 20% increase) and rAAV genome recovery (∼ 20% increase). As a result, the productivity (full capsid generated per liter of AEX column per hour of processing time) of the AEX step increased by ∼34-fold from the baseline AEX batch run to the AEX-WPC-MCC run. It is foreseeable that this AEX-WPC-MCC method could find applications in large-scale rAAV manufacturing processes to improve AEX yield and reduce the cost of goods of rAAV manufacturing.

Simultaneous determination of oxalate and citrate in urine and serum of calcium oxalate kidney stone rats by IP-RP LC-MS/MS

Oxalate and citrate in 24 h urine and serum are considered to be associated with the incidence and recurrence risk of calcium oxalate kidney stones. The quantification of oxalate and citrate contributes to understand the pathological metabolism of kidney stones and guide the early diagnosis and recurrence monitoring. Although simultaneous quantification of oxalate and citrate in urine using liquid chromatography tandem mass spectrometry (LC-MS/MS) have been reported, the optimization of chromatographic column, mobile phase and mass spectrometry (MS) parameters has not been performed. In addition, these is a lack of suitable method for simultaneous detection of oxalate and citrate both in serum and urine. Therefore, we developed a method for the simultaneous determination of oxalate and citrate in urine and serum based on ion-pairing reversed-phase (IP-RP) LC-MS/MS. Herein, five ion-pair reagents, namely, triethanolamine, dimethylbutyl amine, diisopropenyl amine, N,N-dimethylcyclohexylamine and tripropylamine, and three ion-pairing reagent (IPR) buffers, namely, acetic acid, hexafluoro-2-isopropanol, and hexafluoro-2-methyl-2-propanol, were compared in regard to their chromatographic peak abundance and separation of oxalate and citrate. Moreover, MS parameters and the multiple reaction monitoring (MRM) conditions were also evaluated and optimized to obtain the maximum peak abundance. After that, the method was validated in the linear range of 0.25–1000 µM, and the correlation coefficient was ≥ 0.99. The precision and accuracy were < 14.70% and < 19.73%, respectively. The extraction recovery was 80.53–108.79%, and the matrix effect was < 8.79%. The quality control samples were stable at room temperature for 4 h, 4 °C for 24 h, and for 3 freeze–thaw cycles. Finally, this method was applied to the determination of oxalate and citrate in the serum and urine of rats with calcium oxalate kidney stones. The establishment of a stable and effective oxalate and citrate detection method is conducive to the diagnosis and monitoring of kidney stones.

Investigation into reversed-phase chromatography peptide separation systems Part IV: Characterisation of mobile phase selectivity differences

The differentiation of mobile phase compositions between sub-classes which exhibit distinct chromatographic selectivity (i.e. termed characterisation) towards a range of peptide probes with diverse functionality and hence the possibility for multi-modal retention mechanisms has been undertaken. Due to the complexity of peptide retention mechanisms in given mobile phase conditions, no attempt has been made to explain these, instead mobile phases have simply been classified into distinct groups with an aim of identifying those yielding differing selectivities for use in strategic method development roadmaps for the analysis of peptide mixtures.The selectivity differences between nine synthetic peptides (fragments of [Ile27]-Bovine GLP-2) were used to assess how fifty-one RPC mobile phase compositions of differing pH (range 1.8 – 7.8), salt types, ionic strengths, ion-pair reagents and chaotropic / kosmotropic additives affected chromatographic selectivity on a new generation C18 stationary phase (Ascentis Express C18). The mobile phase compositions consisted of commonly used and novel UV or MS compatible additives. The chemometric tool of Principal Component Analysis (PCA) was used to visualise the differences in selectivity generated between the various mobile phases evaluated. The results highlight the importance of screening numerous mobile phases of differing pH, ion-pair reagents and ionic strength in order to maximise the probability of achieving separation of all the peptides of interest within a complex mixture. PCA permitted a ranking of the relative importance of the various mobile phase parameters evaluated. The concept of using this approach was proven in the analysis of a sample of Bovine GLP-2 (1-15) containing synthesis related impurities.Mobile phases with high ionic strength were demonstrated to be crucial for the generation of symmetrical peaks. The observations made on the C18 phase were compared on three additional stationary phases (i.e. alkyl amide, fluorophenyl and biphenyl), which had previously been shown to possess large selectivity differences towards these peptides, on a limited sub-set of mobile phases. With the exception of the ion-pair reagent, similar trends were obtained for the C18, fluorophenyl and biphenyl phases intimating the applicability of these findings to the vast majority of RPC columns (i.e. neutral or weakly polar in character) which are suitable for the analysis of peptides. The conclusions were not relevant for columns with a more disparate nature (i.e. containing a high degree of positive charge).

Quantitative-Profiling Method of Serum Steroid Hormones by Hydroxylamine-Derivatization HPLC\u2013MS

A sensitive and rapid high performance liquid chromatography–mass spectrometry (HPLC–MS) method was developed and validated for simultaneous quantification of ten steroid hormones, including estrogens, androgens, progesterones, and corticosteroids four classes of steroids. The following ten steroid hormones were analyzed: progesterone, 21-deoxycortisol, estrone, 4-androstenedione, testosterone, dihydro-testosterone, androstenone, dehydroepiandrosterone, corticosterone and cortisone. Stable deuterated isotopes were used as internal standards for quantification. Sample preparation with and without derivatization were performed after liquid–liquid extraction, and the corresponding results were compared according to sensitivity and selectivity. Hydroxylamine derivatization was found to improve the ionization efficiency of the analytes for electrospray ionization MS analysis. The gradient of mobile phase and experimental parameters for HPLC separation were optimized. The lower limits of quantification were in the range of 0.05–5 ng mL−1 with wide linear range for the ten steroid hormones. The intra-day precision < 11.1% and recovery of 84.5–120% with negligible matrix effect were achieved, where within the acceptance limits of the FDA guideline. Total HPLC–MS analysis time was 6 min. This method enables simultaneous quantification of steroids in human serum. It will be helpful for the serum steroid profiling in order to understand various endocrinology diseases.Graphical

Simultaneous optimization of mobile phase composition and pH using retention modeling and experimental design

Chromatographic methods are progressing continuously. Increasing sample complexity and safety expectations lead to higher regulatory demands, hence challenges in liquid chromatography analysis are rising, even today, when faster and faster chromatographic systems are extensively employed and become widely accessible for successful method development.The goal of this study was to investigate the impact of mobile phase influences as important factors of selectivity tuning in method development. This would mitigate mobile phase-related robustness issues throughout the method’s lifecycle.To discover and understand these effects, a new module of chromatographic modeling software DryLab (ver. 4.3.4. beta) was introduced and a special experimental design (DoE) was tested, allowing the simultaneous optimization of solvent-dependent parameters, such as gradient time (tG), ternary eluent composition (tC) and pH, requiring 18 input experiments (2 × 3 × 3 = 18).Additionally, the model creation, using a UPLC system and a narrow bore column (50 × 2.1 mm), the entire experimental work could be finished in 2–3 hours. To demonstrate the applicability of this new design, amlodipine and its related pharmacopoeia impurities (A–H) were subjected to be used in a case study. Predicted vs. Experimental (or Verification) runs showed excellent agreement, average retention time deviations were typically less than 1 s. Modelled robustness testing was also performed, elucidating all important mobile phase and instrument parameters that could influence a method’s lifetime performance. Furthermore, as the in silico robustness testing is the least time consuming part of the method development process, it can be used extensively to evaluate robustness even at the very early part in stage 1 of the Method Life Cycle (MLC).

Simultaneous determination of neurotoxic shellfish toxins (brevetoxins) in commercial shellfish by liquid chromatography tandem mass spectrometry

An analytical method using liquid chromatography tandem mass spectrometry has been developed for the determination of neurotoxic shellfish poisoning (NSP) toxins (brevetoxins) in shellfish. The tested parameters included extraction solvent, clean-up method, mobile phase and mass spectrometric detection parameters including multiple reaction monitoring (MRM) conditions. The toxins were extracted with 80% methanol in water and cleaned up using Strata-X SPE cartridges. Chromatographic separation of brevetoxins (PbTx-1, PbTx-2 and PbTx-3) was carried out on a C18 column (100 mm × 2.1 mm, 2.6 μm) with gradient elution. The developed method was validated according to the requirements of SANTE 11945/2015. The average recoveries of the brevetoxins ranged from 75.9% to 105.8%, the intra-day precisions were in the range of 0.4–8.6%, the inter-day precisions were in the range of 0.7–6.3% and the limits of quantification (LOQs) were 25 μg/kg for each toxin. The validated method was successfully applied to real shellfish samples, including mussel, oyster and blood cockle. Overall, the method has shown excellent sensitivity and reproducibility, and it could be used as a reference method for the monitoring of individual brevetoxins in shellfish samples.

Influence of the mobile phase and molecular structure parameters on the retention behavior of protonated basic solutes in chaotropic chromatography

In this study, we present novel insights into the pH-dependent retention behavior of protonated basic solutes in chaotropic chromatography. To this end, two sets of experiments were performed to distinguish between mobile phase pH and ionic strength effects. In the first set, the ionic strength (I) was varied with the concentration of NaPF6 and additives that adjusted the mobile phase pH, while in the second set, I was kept constant by adding the appropriate amount of NaCl. In each set, the retention behavior of 13 analytes was qualitatively examined in 21 chromatographic systems, which were defined by the NaPF6 concentration in their aqueous phases (1–50mM) and the pH of their mobile phases (2, 3 or 4); the acetonitrile content was fixed at 40%. The addition of NaCl significantly reduced the differences among retention factors at studied pH values due to the effect of the Na+ ions on PF6−adsorption to the stationary phase and the magnitude of the consequential development of the surface potential. A quantitative description of the observed phenomenon was obtained by an extended thermodynamic approach. The contribution of ion-pair formation in the stationary phase to the retention of the solutes was confirmed across models at the studied pH values in the set with varying I. In the systems with a constant I, the shielding effect of the Na+ ions on the surface charge lowered the attractive surface potential and diminished the aforementioned interactions and hence the effect of the mobile phase pH on analyte retention. Eventually, we developed a readily interpretable empirical retention model that simultaneously takes into account analyte molecular structures and the most relevant chromatographic factors. Its coefficients have clear physical meaning, and owing to its good predictive capabilities, the model could be successfully used to clarify the contributions of analyte molecular structures and chromatographic factors to the specific processes underlying separation in chaotropic chromatography.

Determination of urazamide in pharmaceutical preparation with room temperature ionic liquid.

A high performance liquid chromatographic method was developed and validated for the determination of urazamide in pharmaceutical preparation with novel green aqueous mobile phase modified with room temperature ionic liquids (RTILs). 1-Ethyl-3-methyl-imidazolium tetrafluoroborate ([EMIM][BF4]) was selected as a mobile phase additive to improve retention and avoid baseline disturbances at t0. Various mobile phase parameters such as cation moiety, chaotropic anion moiety, pH and concentration of RTILs were optimized to determine urazamide at the proper retention time. The assay was validated according to International Conference on Harmonization guidelines. The linearity of the calibration curve was good (r2>0.999). Intra-day precision varied between 0.50 and 1.23%. Relative standard deviations of inter-day precision ranged between 1.07 and 1.66%. Recoveries in tablets ranged between 99.7 and 101.2% and it was successfully applied to determine urazamide in pharmaceutical preparations.

Development and validation of an accurate and sensitive LC-ESI-MS/MS method for the simultaneous determination of paralytic shellfish poisoning toxins in shellfish and tunicate

An analytical method using liquid chromatography-tandem mass spectrometry has been developed for the determination of 5 paralytic shellfish poisoning (PSP) toxins (saxitoxin, gonyautoxin1-4) in shellfish and tunicate. The tested parameters included extraction solvent, clean-up method, mobile phase and mass spectrometric detection parameters including multiple reaction monitoring (MRM) conditions. The PSP toxins were extracted with 0.1 N hydrochloric acid and cleaned up using C18 SPE cartridges. Chromatographic separation of the 5 PSP toxins was carried out on a TSK-gel Amide-80 column with gradient elution. The developed method was validated according to the requirements of SANCO 12571/2013. The average recoveries of the PSP toxins ranged from 82.7% to 110.7%, the intra-day precisions were in the range of 0.3–10.9%, the inter-day precisions were in the range of 0.8–10.0% and the limits of quantification (LOQs) were in the range of 25–100 μg/kg, depending on the analytes and matrices. The validated method was successfully applied for the determination of 5 PSP toxins in real shellfish and tunicate samples, including mussel, oyster, ark shell, sea squirt and styela clava.

Chiral ligand-exchange chromatography with Cinchona alkaloids. Exploring experimental conditions for enantioseparation of α-amino acids

The natural Cinchona alkaloid quinidine as chiral selector in chiral ligand-exchange chromatography was systematically studied. Chromatographic conditions for enantioseparation of twenty α-amino acids were first time studied by changing mobile phase parameters such as pH, concentration of organic solvent, type of salt, ligand to metal ratio and also column temperature. Maximum retention and enantioselectivity factors were observed at the region close to pH=8, since the tertiary amine (the quinuclidinic nitrogen) of the quinidine is protonated only in a small degree, and therefore is available for the chelate formation. Additionally at this pH value there is no other competing ligand for complex the metallic cation. The thermodynamic transfer parameters of the enantiomers from the mobile to the stationary phase from van't Hoff plots within the range of 10–35°C were estimated. Thus, the differences in the transfer enthalpy Δ(ΔH), and transfer entropy Δ(ΔS) enabled an investigation of the origin of the differences in interaction energies Δ(ΔG). Finally, the molecular geometry of the formed diastereomeric complexes was modelled and energetic differences between both compounds were calculated by a semi empirical method.

Enantioseparation and determination of the chiral phenylpyrazole insecticide ethiprole in agricultural and environmental samples and its enantioselective degradation in soil

An effective method for the enantioselective determination of ethiprole enantiomers in agricultural and environmental samples was developed. The effects of solvent extraction, mobile phase and thermodynamic parameters for chiral recognition were fully investigated. Complete enantioseparation of the ethiprole enantiomers was achieved on a Lux Cellulose-2 column. The stereochemical structures of ethiprole enantiomers were also determined, and (R)-(+)-ethiprole was first eluted. The average recoveries were 82.7–104.9% with intra-day RSD of 1.7–8.2% in soil, cucumber, spinach, tomato, apple and peach under optimal conditions. Good linearity (R2≥0.9991) was obtained for all the matrix calibration curves within a range of 0.1 to 10mgL−1. The limits of detection for both enantiomers were estimated to be 0.008mgkg−1 in soil, cucumber, spinach and tomato and 0.012mgkg−1 in apple and peach, which were lower than the maximum residue levels established in Japan. The results indicate that the proposed method is convenient and reliable for the enantioselective detection of ethiprole in agricultural and environmental samples. The behavior of ethiprole in soil was studied under field conditions and the enantioselective degradation was observed with enantiomer fraction values varying from 0.494 to 0.884 during the experiment. The (R)-(+)-ethiprole (t1/2=11.6d) degraded faster than (S)-(−)-ethiprole (t1/2=34.7d). This report is the first describe a chiral analytical method and enantioselective behavior of ethiprole, and these results should be extremely useful for the risk evaluation of ethiprole in food and environmental safety.

Strategy Approach for Direct Enantioseparation of Hyoscyamine Sulfate and Zopiclone on a Chiral αl‐Acid Glycoprotein Column and Determination of Their Eutomers: Thermodynamic Study of Complexation

Rapid and simple isocratic high-performance liquid chromatographic methods with UV detection were developed and validated for the direct resolution of racemic mixtures of hyoscyamine sulfate and zopiclone. The method involved the use of αl -acid glycoprotein (AGP) as chiral stationary phase. The stereochemical separation factor (ά) and the stereochemical resolution factor (Rs ) obtained were 1.29 and 1.60 for hyoscyamine sulfate and 1.47 and 2.45 for zopiclone, respectively. The method was used for determination of chiral switching (eutomer) isomers: S-hyoscyamine sulfate and eszopiclone. Several mobile phase parameters were investigated for controlling enantioselective retention and resolution on the chiral AGP column. The influence of mobile phase, concentration and type of uncharged organic modifier, ionic strength, and column temperature on enantioselectivity were studied. Calibration curves were linear in the ranges of 1-10 µg mL(-1) and 0.5-5 µg mL(-1) for S-hyoscyamine sulfate and eszopiclone, respectively. The method is specific and sensitive, with lower limits of detection and quantifications of 0.156, 0.515 and 0.106, 0.349 for S-hyoscyamine sulfate and eszopiclone, respectively. The method was used to identify quantitatively the enantiomers profile of the racemic mixtures of the studied drugs in their pharmaceutical preparations. Thermodynamic studies were performed to calculate the enthalpic ΔH and entropic ΔS terms. The results showed that enantiomer separation of the studied drugs were an enthalpic process.

Quantitative isomer-specific N-glycan fingerprinting using isotope coded labeling and high performance liquid chromatography–electrospray ionization-mass spectrometry with graphitic carbon stationary phase

Glycan reductive isotope labeling (GRIL) using 12C6-/13C6-aniline as labeling reagent is reported with the aim of quantitative N-glycan fingerprinting. Porous graphitized carbon (PGC) as stationary phase in capillary scale HPLC coupled to electrospray mass spectrometry with time of flight analyzer was applied for the determination of labeled N-glycans released from glycoproteins. The main benefit of using stable isotope-coding in the context of comparative glycomics lies in the improved accuracy and precision of the quantitative analysis in combined samples and in the potential of correcting for structure-dependent incomplete enzymatic release of oligosaccharides when comparing identical target proteins. The method was validated with respect to mobile phase parameters, reproducibility, accuracy, linearity and limit of detection/quantification (LOD/LOQ) using test glycoproteins. It is shown that the developed method is capable of determining relative amounts of N-glycans (including isomers) comparing two samples in one single HPLC-MS run. The analytical potential and usefulness of GRIL in combination with PGC-ESI-TOF-MS is demonstrated comparing glycosylation in human monoclonal antibodies produced in Chinese hamster ovary cells (CHO) and hybridoma cell lines.

Assessing Drug-Excipient Interactions in the Formulation of Isoniazid Tablets

A suitable and efficient high-performance liquid chromatography (HPLC) method was developed for the simultaneous determination of tuberculostatic isoniazid and its related impurities, isonicotinic acid and isonicotinamide, in oral solid dosage forms. We studied the influence on chromatographic separation of mobile phase parameters, such as pH, ion pairing and ionic strength, as well as column bonded phase and brands. The use of buffer solution pH 6.8:acetonitrile 96:4 (v/v) at 0.8 mL min-1, C18 column (250 × 4.6 mm, 5 µm), allowed proper separation of these ionizable analytes without the need of ion pairing agents. The limit of quantification of the method (0.1 µg mL-1) was suitable for the analysis of impurities. A drug-excipient compatibility study was carried out with the anaytical method proposed in this work and addressed frequent excipients used in isoniazid coated tablets. The dye FD&C blue 2 lake promoted the highest degradation of the tuberculostatic agent.

Enantioseparation of N-derivatized amino acids by micro-liquid chromatography using carbamoylated quinidine functionalized monolithic stationary phase

In order to obtain satisfactory column permeability, efficiency and selectivity for micro-HPLC, a capillary monolithic column containing O-9-[2-(methacryloyloxy)-ethylcarbamoyl]-10,11-dihydroquinidine (MQD) as chiral selector was re-optimized. The monolithic column was used to successfully enantioresolve a wide range of N-derivatized amino acids including alanine, leucine, methionine, threonine, phenylalanine, valine, serine, isoleucine, tryptophan, and cysteine. The influence of mobile phase parameters, such as the organic solvent type and concentration, the apparent pH, and buffer concentration, on retention and enantioseparation of N-derivatized amino acids has been investigated. 3,5-dinitrobenzoyl-amino acids and 3,5-dichlorobenzoyl-amino acids were resolved into enantiomers with exceptionally high selectivity and resolution. The chemoselectivity of the monolithic column for a multicomponent mixture of N-derivatized amino acids was also investigated. A mixture of three pairs of 3,5-dichlorobenzoyl-amino acids could be fully resolved in 22.5min.

Separation of xanthines in hydro-organic and polar-organic elution modes on a titania stationary phase

Hydrophilic interaction LC was investigated in hydro-organic and nonaqueous elution modes on a titania column by using a set of N-methyl xanthines as neutral polar probes. To get information regarding the mechanisms that are behind the discrimination of these analytes in hydrophilic interaction, we focused our study on the type and amount of organic modifier as a critical yet rarely explored mobile phase parameter. Several alcohols such as methanol, ethanol, and isopropanol were studied as substitutes to acetonitrile in hydro-organic elution mode. Compared to silica, the investigation of the eluotropic series of these alcohols on titania highlighted a different implication in the retention mechanism of the xanthine derivatives. At low amounts of protic solvents, the adsorption mainly characterized the retention of analytes on bare silica; whereas mixed interactions including adsorption and ligand exchange were identified on native titania. To investigate the peculiar behavior of alcohols on the metal oxide, methanol, ethanol, and ethylene glycol were tested in replacement of water in polar-organic elution mode. Distinctive effects on the chromatographic retention and selectivity of xanthines were noticed for the dihydric alcohol, which was found to be a stronger eluting component than water on titania.

Simultaneous analysis of iodate, iodide, bromate and bromide by ion chromatography with ultraviolet detection

An analytical method of ion chromatography with ultraviolet detection has been developed and applied for the simultaneous determination of iodate, iodide, bromate and bromide. The separation was performed on a quaternary ammonium type anion exchange column with citric acid and acetonitrile as mobile phase. The effects of the detection wavelength, the kind and concentration of the mobile phase and other parameters on separation and detection of the four ions were investigated. The retention rules were studied and the chromatographic conditions were optimized. Under the conditions of 210 nm as detection wavelength, 0.9 mL/min as flow rate, 40 degrees C as column temperature, and 1.0 mmol/L citric acid-acetonitrile (85:15, v/ v; pH 5.0) as mobile phase, the four ions were completely separated and the system peaks and other common anions didn't interfere with the determination. The detection limits of the four ions (S/N = 3) were 0.07-0.16 mg/L. The relative standard deviations of the retention times and peak areas obtained by determining samples five times continuously were below 1%. The spiked recoveries of the four anions were from 98.0% to 102%. This method has been successfully used to determine ionic liquids synthesized by chemistry laboratory and underground water samples. The results were accurate and reliable.

Determination of Thiamine in Pharmaceutical Preparations by Reverse Phase Liquid Chromatography Without Use of Organic Solvent

A novel green aqueous mobile phase modified with room temperature ionic liquids (RTILs) was employed in the absence of volatile organic solvents or ion-pairing reagents to analyze thiamine, a very polar compound, by reverse phase high performance liquid chromatography (RP-HPLC). Due to its strongly hydrophilic nature, thiamine was eluted near the column dead time (t0) using a mobile phase without adding RTILs or ion-pairing reagents, even if a 100% aqueous mobile phase, which has weak elution power under reverse phase conditions, was used. Thus, 1-ethyl-3-methyl-imidazolium hexafluorophosphate ([EMIM][PF6]), which has the strongest chaotropic effect, was selected as a mobile phase additive to improve retention and avoid baseline disturbances at t0. Various mobile phase parameters such as cation moiety, chaotropic anion moiety, pH and concentration of RTILs were optimized to determine thiamine at the proper retention time. Method validation was performed to assess linearity, intra- and inter-day accuracy and precision, recovery and repeatability; all results were found to be satisfactory. The developed method was also compared to the current official United States Pharmacopoeia (USP) and Korean Pharmacopoeia (KP) methods using an organic mobile phase containing an ionpairing reagent by means of evaluating various chromatographic parameters such as the capacity factor, theoretical plate number, peak asymmetry and tailing factor. The results indicated that the proposed method exhibited better efficiency of thiamine analysis than the official methods, and it was successfully applied to quantify thiamine in pharmaceutical preparations.