Determination of glutathione and cysteine in yeasts by hydrophilic interaction liquid chromatography followed by on‐line postcolumn derivatization

In the present study, we report a new method for the determination of two primary thiols, cysteine (CYS) and glutathione (GSH), by hydrophilic interaction LC. The polar analytes are separated isocratically using a mobile phase consisting of 65% acetonitrile/35% ammonium acetate (15 mmol/L, pH 2.0) and are detected at 285 nm following on‐line postcolumn derivatization by the thiol‐selective reagent methyl propiolate. The main figures of merit included linearity in the range of 5–200 μmol/L and an LOD 0.6 μmol/L for both compounds. The absence of matrix effect allowed the determination of CYS and GSH in various yeast samples. GSH was present in most of the samples at levels ranging between 0.9 and 3.1 mg/g, whereas CYS was determined in only one sample at a significantly lower concentration. In terms of accuracy, the percent recoveries ranged between 91.2 and 105.6% for GSH, and 91.6 and 106.9% for CYS.     

In‐column preparation of a brush‐type chiral stationary phase using click chemistry and a silica monolith

Brush‐type chiral stationary phases (CSP) have been prepared both from a silica monolith and, separately, from 10 μm porous silica beads via a process of in‐column modification including attachment of the chiral selector via copper‐catalyzed azide–alkyne cycloaddition. Azide functionalities were first introduced on the pore surface of each type of support by reaction with 3‐(azidopropyl)trimethoxysilane, followed by immobilization of a proline‐derived chiral selector containing an alkyne moiety. This functionalization reaction was carried out in dimethylformamide (DMF) in the presence of catalytic amounts of copper (I) iodide. The separation performance of these triazole linked stationary phases was demonstrated in enantioseparations of four model analytes, which afforded separation factors as high as 11.4.     

Silica‐based zwitterionic monolithic stationary phase for separation of neutral and ionized solutes using pressurized CEC

A porous zwitterionic monolith was prepared by in situ covalent attachment of lysine to a γ‐glycidoxypropyltrimethosysilane‐modified silica monolith. The prepared column was used to perform neutral and ionized solutes separations by pressurized (pCEC). Due to the zwitterionic nature of the resulting stationary phase, the monolithic column provided both electrostatic attraction and repulsion sites for electrochromatographic retention for ionized solutes. Separation of several nucleotides was investigated on the monolithic column. It was shown that the nucleotides could be separated based on hydrophilic and electrostatic interactions between the stationary phase and analyte. Besides, the separation property of the zwitterionic silica monolith was compared with the use of diamine‐bonded silica monolith as stationary phase. As expected, the lysine monolith exhibited a lower retention for the five nucleotides, which was due to the dissociation of the external carboxylic acid groups, leading to electrostatic repulsion with negatively charged solutes. Under the same experimental conditions, separation of the five nucleotides on the zwitterionic column was in less than 8 min, while that on the diamine column was in approximately 60 min.     

LC of sulfonamide residues in poultry muscle and eggs extracts using fluorescence pre‐column derivatization and monolithic silica column

A new, rapid, sensitive and selective HPLC method with fluorescence detection is described for the simultaneous determination of 12 sulfonamides, in the presence of putrescine as internal standard, after pre‐column derivatization with fluorescamine. The drugs were separated on a Chromolith Performance RP‐18 column (100×4.6 mm), using a gradient elution with a binary mobile phase of methanol/0.05 M acetate buffer (pH 3.4). Linearity of derivatization was obtained for concentrations from 3.0 to 300 μg/L in standard solutions. The whole procedure was evaluated and fully validated, according to the European Union Decision 2002/657/EC, for the determination of sulfonamides in turkey muscle and hen eggs following SPE. The LODs varied from 2 to 17 μg/kg in turkey and 2 to 15 μg/kg in egg samples. The average recoveries ranged between 96.9–108.6% in turkey muscle and 96.0–108.4% in egg samples, respectively.     

A rapid HPLC method for the determination of carboxylic acids in human urine using a monolithic column

A rapid HPLC method for the determination of carboxylic acids in urine samples using a Chromolith Performance RP/18e 100/4.6 with Chromolith Guard Cartridge RP/18e 10/4.6 (Merck KgaA, Darmstadt, Germany) was developed. The method facilitates the simultaneous determination of aromatic hydrocarbon metabolites mandelic acid (MA) and phenylglyoxylic acid (PGA) from styrene and ethylbenzene, hippuric acid (HA) from toluene and 2-, 3-, 4-methylhippuric acids (MHA) from xylene. 3-Hydroxybenzoic acid (3-HBA) was used as internal standard. A chromatographic run is completed within less than 5 min for styrene, ethylbenzene and toluene metabolites, and within 10 min for xylene metabolites. The detection limits are 9 mg L^–1 urine for MA, 1.25 mg L^–1 urine for PGA, 4.9 mg L^–1 urine for HA, 22 mg L^–1 urine for 2-MHA, and 18.5 mg L^–1 urine for 3-MHA.No significant differences of the MA, PGA and HA concentrations in human urine samples obtained by HPLC chromatography on LiChrosorb RP 18 and on Chromolith RP/18e columns were found. The results were evaluated by using ANOVA.Abbreviations MA mandelic acid - PGA phenylglyoxylic acid - HA hippuric acid - MHA methylhippuric acid - 3-HBA 3-hydroxybenzoic acid - ANOVA analyses of variance - GC gas chromatography - HPLC high-performance liquid chromatography     

Determination of tadalafil in pharmaceutical preparation by HPLC using monolithic silica column

The simple, reliable and reproducible HPLC and extraction methods were developed for the analysis of tadalafil in pharmaceutical preparation. The column used was monolithic silica column, Chromolith Performance RP-18e (100 mm × 4.6 mm, i.d.). The mobile phase used was phosphate buffer (100 mM, pH 3.0)-acetonitrile (80:20, v/v) at the flow rate of 5 mL min⁻¹ with UV detection at 230 nm at ambient temperature. Extraction of tadalafil from tablet was carried out using methanol. Linearity was observed in the concentration range from 100 to 5000 ng mL⁻¹ for tadalafil with a correlation coefficient (R²) 0.9999 and 100 ng mL⁻¹ as the limit of detection. The values of linearity range, correlation coefficient (R²) and limit of detection were 50-5000 ng mL⁻¹, 0.9999-50 ng mL⁻¹, respectively for sildenafil. Parameters of validation prove the precision of the method and its applicability for the determination of tadalafil in pharmaceutical tablet formulation. The method is suitable for high throughput analysis of the drug.     

Optimization of St John's Wort flavonoid separation by reversed phase liquid chromatography on a silica-based monolithic column

Summary An optimization procedure for the separation of flavonoids from St John's Wort by reversed-phase high-performance liquid chromatography on monolithic stationary phase is described. Three-component mobile phase systems are studied using experimental design methodology. The starting experimental domain is a triangle, each vertex of which is a pure component. From preliminary isocratic experiments, truncations in the domain are performed leading to a quadrilateral shape working domain with no symmetry. To cope with both separation and analysis time, desirability functions are used. Optimal conditions are finally given by binary systems and the four flavonoids are separated in less than seven minutes.     

Rapid analysis of pyrethroids in whole urine by high-performance liquid chromatography using a monolithic column and off-line preconcentration in a restricted access material cartridge

A rapid high-performance liquid chromatography (HPLC) method using a monolithic column with UV detection at 238 nm was developed for the determination of fenpropathrin, betacyfluthrin, deltamethrin, and permethrin (cis and trans isomers) in whole urine. The method is based on the use of a monolithic chromatographic column and a restricted access material (RAM) cartridge for sample preparation. The mobile phase was water/acetonitrile (42:58 v/v), the flow rate was 3 mL min^–1, and chromatographic separation was carried out in 10 min. The separation of cis and trans isomers of permethrin was also possible under the above-mentioned conditions. Detection limits in reconstituted whole urine samples were between 0.9 g L^–1 for betacyfluthrin and 4.4 g L^–1 for fenpropathrin and trans-permethrin. Recoveries for urine samples spiked with different amounts of pyrethroids (between 19 g L^–1 and 75 g L^–1) were in the 70±6 to 90±7% range.     

Trace analysis of rimantadine in human urine after dispersive liquid liquid microextraction followed by liquid chromatography–post column derivatization

The first dispersive liquid liquid microextraction scheme followed by liquid chromatography‐post column derivatization for the determination of the antiviral drug rimantadine in urine samples is demonstrated. The effect of the type and volume of organic extraction solvent, type and volume of disperser solvent, sample pH, ionic strength, extraction time, and centrifugation speed on the extraction efficiency were studied. Rimantadine and the internal standard (amantadine) were chromatographed using a reversed phase monolithic stationary phase with a mixture of equal volumes of methanol and phosphate buffer (pH = 3) as mobile phase. On‐line post‐column derivatization of the analyte was performed using a “two‐stream” manifold with o‐phthalaldehyde and N‐acetyl‐cysteine at alkaline medium. Under the optimized extraction conditions, the enrichment factor of rimantadine was 58. The linear range was 5–100 µg/L with correlation coefficient r of 0.9984 while the limit of detection achieved was 0.5 µg/L. The within‐day and between‐day precision for the tested concentration levels were less than 14.3% and the mean recoveries obtained from the spiked samples were ranged between 87.5 and 113.9%. The main advantages of the proposed method are the simplicity of operation, rapidity, low cost, and low limit of detection of the analyte.     

Retention behaviour of beta-blockers in HPLC using a monolithic column

The chromatographic behaviour of a new HPLC-stationary phase (Chromolith RP-18e) is described for separation of beta-blockers. The effects of the different chromatographic conditions (buffer system, pH value, content of organic modifier, injection volume and flow rate) on the separation behaviour were studied. At higher flow rates the peaks seemed to be more symmetrical than at lower flow rates. The use of buffer or salt in the mobile phase for this separation is found to be very essential and the counter-anion type of this buffer or salt significantly affected the retention behaviour of beta-blockers while the cation type did not play the same important role.     

Development and validation of a high‐performance liquid chromatography method for determination of lisinopril in human plasma by magnetic solid‐phase extraction and pre‐column derivatization

A sensitive, reliable and simple HPLC method was developed for the determination of lisinopril in human plasma. The method consists of extraction and clean‐up steps based on magnetic solid‐phase extraction and pre‐column derivatization with a fluorescent reagent. The mobile phase consisted of a mixture of methanol–sodium dihydrogen phosphate (pH 3.0; 0.005 m ; 75:25, v/v). The flow rate was set at 0.7 mL/min. Fluorescence detection was performed at 470nm excitation and 530nm emission wavelengths. Total chromatography run time was 5 min. The average extraction recovery of lisinopril and fluvoxamine (internal standard) was ≥82.8%. The limits of detection and quantification were determined as 1 and 3 ng/mL respectively. The method exhibited a linear calibration line over the concentration range of 3–1000 ng/mL with coefficient of determination (r²) of ≥0.98. The within‐run and between‐run precisions were satisfactory with values of CV of 1.8–12.8% (accuracy from 99.2 to 94.7%) and 2.4–13.7% (accuracy from 99.5 to 92.2%), respectively. These developments led to considerable improvement in method sensitivity and reliability. The method was validated according to the US Food and Drug Administration guidelines. Therefore, it can be considered as a suitable method for determination of lisinopril in plasma samples.     

HPLC microdetermination of flurbiprofen enantiomers in plasma with a glycopeptide-type chiral stationary phase column

A rapid and stereospecific HPLC micromethod to quantify flurbiprofen enantiomers was developed. Both flurbiprofen enantiomers and indomethacin, used as internal standard, were extracted with methylene chloride from 100 microL of acidified plasma. The resolution of the R- and S-forms was performed on a bonded vancomycin chiral stationary phase (Chirobiotic V) with 20% of tetrahydrofuran in ammonium nitrate (100 mM, pH 5) as mobile phase. Calibration curves were linear in the range 0.5-10 microg/mL for both enantiomers. A good accuracy (< or = 5%) was obtained for all quality controls, with intra-day and inter-day variation coefficients equal or less than 7.7%. Recovery of both enantiomers was found in the range 77.4-86.3%. The lower limit of quantitation was 0.25 microg/mL for both enantiomers, without interference of endogenous components. This validated micromethod has been successfully applied for quantifying R- flurbiprofen and S- flurbiprofen in rat plasma.     

Multiple monolithic fiber solid‐phase microextraction based on a polymeric ionic liquid with high‐performance liquid chromatography for the determination of steroid sex hormones in water and urine

The development of a simple and sensitive analytical approach that combines multiple monolithic fiber solid‐phase microextraction with liquid desorption followed by high‐performance liquid chromatography with diode array detection is proposed for the determination of trace levels of seven steroid sex hormones (estriol, 17β‐estradiol, testosterone, ethinylestradiol, estrone, progesterone and mestranol) in water and urine matrices. To extract the target analytes effectively, multiple monolithic fiber solid‐phase microextraction based on a polymeric ionic liquid was used to concentrate hormones. Several key extraction parameters including desorption solvent, extraction and desorption time, pH value and ionic strength in sample matrix were investigated in detail. Under the optimal experimental conditions, the limits of detection were found to be in the range of 0.027–0.12 μg/L. The linear range was 0.10–200 μg/L for 17β‐estradiol, 0.25–200 μg/L estriol, ethinylestradiol and estrone, and 0.50–200 μg/L for the other hormones. Satisfactory linearities were achieved for analytes with the correlation coefficients above 0.99. Acceptable method reproducibility was achieved by evaluating the repeatability and intermediate precision with relative standard deviations of both less than 8%. The enrichment factors ranged from 54‐ to 74‐fold. Finally, the proposed method was successfully applied to the analysis of steroid sex hormones in environmental water samples and human urines with spiking recoveries ranged from 75.6 to 116%.     

On-line concentration of neutral and charged species in capillary electrochromatography with a methacrylate-based monolithic stationary phase

A method involving self-concentration, on-column enrichment and field-amplified sample stacking for on-line concentration in capillary electrochromatography with a polymer monolithic column is presented. Since monolithic columns eliminate the frit fabrication and the problems associated with frits, the experimental conditions could be more flexibly adjusted to obtain higher concentration factor in comparison with conventional particulate packed columns. With self-concentration effect, the detection sensitivity of benzene and hexylbenzene is improved by a factor of 4 and 8, respectively. With on-column enrichment and ultralong injection, improvement as high as 22,000 times in detection sensitivity of benzoin is achieved. Furthermore, a combination of the three above-mentioned methods yields up to a 24,000-fold improvement in detection sensitivity for caffeine, a charged compound. Parameters affecting the efficiency of on-line concentration are investigated systematically. In addition, equations describing on-line concentration process are deduced.     

Preparation of a monolithic column with a mixed‐mode stationary phase of reversed‐phase/hydrophilic interaction for capillary liquid chromatography

A monolithic capillary column with a mixed‐mode stationary phase of reversed‐phase/hydrophilic interaction chromatography was prepared for capillary liquid chromatography. The monolith was created by an in‐situ copolymerization of a homemade monomer N,N‐dimethyl‐N‐acryloxyundecyl‐N‐(3‐sulfopropyl) ammonium betaine and a crosslinker pentaerythritol triacrylate in a binary porogen agent consisting of methanol and isopropanol. The functional monomer was designed to have a highly polar zwitterionic sulfobetaine terminal group and a hydrophobic long alkyl chain moiety. The composition of the polymerization solution was systematically optimized to permit the best column performance. The columns were evaluated by using acidic, basic, polar neutral analytes, as well as a set of alkylbenzenes and Triton X100. Very good separations were obtained on the column with the mixed‐mode stationary phase. It was demonstrated that the mixed‐mode stationary phase displayed typic dual retention mechanisms of reversed‐phase/hydrophilic interaction liquid chromatography depending on the content of acetonitrile in the mobile phase. The method for column preparation is reproducible.     

Liquid chromatographic high-throughput analysis of ketamine and its metabolites in human plasma using a monolithic silica column and solid phase extraction

A rapid and sensitive liquid chromatographic (LC) assay was developed for the simultaneous determination of ketamine (KE) and its two main metabolites, namely, norketamine (NK) and dehydronorketamine (DHNK) in human plasma. Each compound together with an internal standard (Labetalol) was extracted from the plasma matrix using solid phase extraction (SPE). The applicability of monolithic LC phases in the field of quantitative bioanalysis has been evaluated. The existing method with UV detection set at 220 nm was successfully transferred from a conventional reversed phase column to a 10 cm × 4.6 mm i.d. monolithic silica column. By simply increasing the mobile phase flow-rate, run times were about six-fold reduced and consumption of mobile phase were about two-fold decreased, while the chromatographic resolution of the analytes remain unaffected. The method was validated over the range 25-2000 ng/mL for KE, 25-1500 ng/mL for NK, and 15-750 ng/mL for DHNK. The method proved to be precise (within-run precision ranges from 2.2 to 7.2% and between-run precision ranges from 3.7 to 8.2%) and accurate (within-run accuracies ranged from 1.3 to 7.2% and between-run accuracies ranged from 1.5 to 8.7%). The mean absolute recoveries were 95.3, 96.9, and 103.9% for KE, NK and DHNK, respectively. The limit of quantitation (LOQ) and limit of detection (LOD) for KE and NK in human plasma were 25 and 12.5 ng/mL, respectively, and for DHNK were 15 and 7.5 ng/mL (S/N = 3). The assay should be suitable for use in routine determination of KE and its metabolites in human plasma.     

Determination of urinary 8‐hydroxy‐2′‐deoxyguanosine by a combination of on‐line molecularly imprinted monolithic solid phase microextraction with high performance liquid chromatography–ultraviolet detection

8‐Hydroxy‐2′‐deoxyguanosine (8‐OHdG) is a sensitive biomarker for DNA oxidative damage. However, its determination in human urine is confounded by trace level and complex matrix. In this study, a new configuration of on‐line solid phase microextraction coupled to high performance liquid chromatography‐ultraviolet detection was established with molecularly imprinted monolithic column as extraction sorbent. The tailor made monolith exhibited high extraction efficiency with the enrichment factor 101.84 for 8‐OHdG owing to its special porous structure and inherent selectivity. Under optimal condition, appreciable sensitivity had been achieved for this incorporation with limit of detection 2.04 nmol/L (S/N = 3) and limit of quantification 7.12 nmol/L (S/N = 10), respectively. Precise determination with wide range linearity (0.007–5.00 μmol/L) afforded a practical alternative in urinary 8‐OHdG analysis and 107 different subjects had been successfully analyzed. This newly developed method embodied useful prospect for the investigation of DNA oxidative damage with less expense, convenient maintenance and ease of operation     

Determination of fluoroquinolone antibiotics in hospital and municipal wastewaters in Coimbra by liquid chromatography with a monolithic column and fluorescence detection

The main goal of this work was determination of residues of the antibiotics ofloxacin (OFLO), norfloxacin (NOR), ciprofloxacin (CIPRO), and enrofloxacin (ENRO) in wastewater samples. The samples, after acidification to pH 4.5 and addition of EDTA, were extracted on an anion-exchange cartridge in tandem with an Oasis HLB cartridge. The LC–FD method, developed in previous studies, was based on application of a monolithic C₁₈ column. The limit of quantification (LOQ) of the method was 250 ng L⁻¹ for OFLO, 25 ng L⁻¹ for NOR and CIPRO, and 50 ng L⁻¹ for ENRO. Mean recovery ranged between 75 and 121% for OFLO, NOR, CIPRO, and ENRO. A total of 14 wastewater samples were analyzed; these were collected from four hospitals and from influent and effluent from a wastewater-treatment plant in Coimbra, Portugal, during spring and autumn. CIPRO was present in all the samples, NOR was detected second most often, followed by OFLO. ENRO was found at concentrations under the LOQ in five hospital samples, and the highest level was found in influent from the WWTP.     

Alkaloid purification using rosin‐based polymer‐bonded silica stationary phase in HPLC

Alkaloids are important natural products that exhibit a wide spectrum of pharmacological activities. To efficiently separate and purify them, a rosin‐based polymer‐bonded silica stationary phase in high‐performance liquid chromatography was synthesized via the surface radical polymerization of ethylene glycol maleic rosinate acrylate and methacrylic acid onto functionalized silica. The stationary phases, columns, optimization of chromatographic conditions for alkaloids, and thermodynamic behavior of the analytes on the column were fully studied. Under the optimized conditions, the prepared column efficiently purified natural camptothecine, caffeine, and evodiamine with the corresponding purities of 92, 96, and 97%. With this work, we have developed an efficient approach to isolate alkaloids and promoted the research on rosin‐based materials in biomedicine and analytical chemistry.     

Determination of intracellular glutathione and cysteine using HPLC with a monolithic column after derivatization with monobromobimane

An optimized high‐performance liquid chromatography (HPLC) method is used to show that, as myoblasts differentiate into multinucleated muscle fibers, there is a shift to a more oxidized cell redox state. The HPLC method incorporated derivatization with monobromobimane for the determination of the reduced (GSH) and oxidized (GSSG) forms of glutathione and the reduced (Cys) and oxidized (CysSS) forms of cysteine. The derivatization was optimized to improve the sensitivity of the approach; the limits of detection for glutathione and cysteine were 3 × 10^?8 and 5 × 10^?8 M , respectively. Copyright © 2009 John Wiley & Sons, Ltd.