Prediction of the chromatographic behaviour for a series of diuretic compounds

Summary The proportion of organic modifier and the pH of the acetonitrile-water mixtures used as mobile phases were optimized in order to separate a group of diuretic compounds covering a wide range of physyco-chemical properties. The Linear Solvation Energy Relationship (LSER) formalism based either on the multiparameter π^*, β and α scales or the single solvent polarity parameterE _T ^N , have been used to predict their chromatographic behaviour as a function of the percentage of acetonitrile in the eluent. Moreover, correlation established between retention and pH of the aqueous-organic mobile phases have been used to predict the chromatographic behaviour of the diuretic compounds studied as a function of the eluent pH. Linear correlation between a function of the eluent pH. Linear correlation between the chromatographic retention and theE _T ^N polarity parameter of mobile phases containing different percentages of organic modifier has been obtained Based on the knowledge of the acid-base dissociation constant the relation between retention and mobile phase pH has also been linearized. These relationship allowed an important reduction of the experimental retention data needed for developing a given separation and a great improvement in chromatographic optimization schemes.     

Optimisation of mobile phase pH in liquid chromatography. Application to fluorimetric detection of series of quinolones

Summary The dissociation constants of several quinolones in a 7% acetonitrile-water mixture are determined. A model describing the effect of pH on the retention of quinolones by an octadecylsilica stationary phase with a 7% acetonitrile-water mobile phase is proposed. The model uses pH values in the mobile phase instead of pH values in water and takes into account the effect of activity coefficients. Fluorescence of quinolones in the mobile phase is studied, and a chromatographic separation with fluorimetric detection is established. Detection limits for the proposed method range from 3 to 12 μg·L⁻¹.     

Determination of phenolic compounds in water samples by on-line solid-phase extraction—supercritical-fluid chromatography with diode-array detection

Summary The eleven Environmental Protection Agency (EPA) priority phenolic compounds have been determined by solid-phase extraction (SPE) coupled on-line to supercritical-fluid chromatography (SFC) with diodearray detection. The variables affecting chromatographic separation were optimized and the analytes were separated at 40 °C in two diol columns connected in series; a gradient of methanol, as modifier, and CO₂ was used as mobile phase. Under these conditions, all the compounds studied were separated to baseline in less than 13 min. PLRP-S and LiChrolut EN were tested as sorbents in a 10×3 mm i.d. laboratory-packed precolumn for solid-phase extraction. An ion-pair reagent, tetrabutylammonium bromide (TBA), was used in the extraction process to increase break-through volumes. The performance of the method was checked with tap and river waters and the pre-concentration of 20 mL of sample in a PLRP-S pre-column enabled phenolic compounds to be determined at low μg L⁻¹ levels with limits of detection ranging between 0.4 and 2 μg L⁻¹. The repeatability and reproducibility between days (n=3) for real samples spiked at 10 μg L⁻¹ were lower than 10%.     

Separation of L-DOPA and four metabolites in plasma using a porous graphitic carbon column in capillary liquid chromatography

Summary A sensitive HPLC method with marbofloxacin (MAR) as internal standard and fluorescence detection is described for the analysis of ofloxacin (OFL) enantiomers in plasma samples. Plasma samples were prepared by adding phosphate buffer (pH 7.4, 0.1m), then extracted with trichloromethane.S-OFL,R-OFL, and the internal standard were separated on a reversed-phase column with water-methanol, 85.5∶14.5, as mobile phase. The concentrations ofS-OFL andR-OFL eluting from the column (retention times 7.5 and 8.7 min, respectively) were monitored by fluorescence detection withλ _ex = 331 andλ _em = 488 nm. The detection and quantitation limits were 10 and 20 ng mL⁻¹, respectively, forS-OFL and 11 and 21 ng mL⁻¹ forR-OFL. Response was linearly related to concentration in the range 10 to 2500 ng mL⁻¹. Recovery was close to 93% for both compounds. The method was applied to determination of the enantiomers of OFL in plasma samples collected during pharmacokinetic studies.     

High-performance liquid chromatographic determination of diltiazem in human plasma after solid-phase and liquid–liquid extraction

A selective, sensitive, and accurate high-performance liquid chromatographic method for determination of diltiazem in plasma samples has been developed and validated. The effects of mobile phase composition, buffer concentration, mobile phase pH, and concentration of organic modifiers on retention of diltiazem and internal standard were investigated. Solid-phase and liquid–liquid extraction were examined and proposed for isolation of the drug and elimination of endogenous plasma interferences. A 5 m Lichrocart Lichrospher 60 RP-select B chromatographic column was used; the mobile phase was acetonitrile–0.025 mol L^–1 KH₂PO₄ (pH 5.5), 35:65 ( v / v) at a flow-rate of 1.5 mL min^–1. The detection wavelength was 215 nm. The calibration plots were linear in the concentration range 20.0–500.0 ng mL^–1. The method has been implemented to monitor diltiazem levels in patient samples.     

Simultaneous determination of difloxacin and its primary metabolite sarafloxacin in rabbit plasma

Summary An HPLC method with fluorescence detection is presented for the analysis of difloxacin (DIF) and sarafloxacin (SAR) in rabbit plasma using norfloxacin (NOR) as internal standard (Figure 1). Plasma sample preparations were carried out by adding phosphate buffer (pH 7.4, 0.1 M), followed by extraction with trichloromethane. Fluoroquinolones were separated on a reversed-phase column using an aqueous phosphate solution-acetonitrile (82:18) mobile phase. The concentrations of NOR, SAR and DIF eluting off the column, with retention times of 2.16, 5.60 and 6.20, respectively, were monitored by fluorescence detection atλ _ex 338 andλ _em 425 nm. The quantitation limit was 12 ng mL⁻¹ for SAR and DIF. Standard curves were linearly related to concentration in the range from 1 to 1500 ng mL⁻¹. Recovery was determined as 76% and 70% for SAR and DIF, respectively. Inter-and intraassay coefficients of variation were less than 6% for all compounds.     

Determination of rutin and forsythin in fruit ofForsythia suspensa(Thunb.) Vahl by capillary electrophoresis-electrochemical detection

Summary Capillary electrophoresis-amperometric detection is evaluated for simultaneous determination of rutin and forsythin. The cyclic voltammogram, hydrodynamic voltammogram, effect of pH, buffer concentration and SDS, and percent organic modifier on separation and detection were studied. Conditions were optimized as follows: 1.2 V detection potential; separation at 12 kV; 5 s at 15 kV for sample injection time and sample injection voltage; mobile phase 20 mM boric acid buffer; pH 8.4, containing 40 mM SDS and 10% (v/v) acetontrile. The method gave low detection limit as 0.001 mg mL⁻¹ and 0.0005 mg mL⁻¹ (S/N=3), wide linear range 0.005–0.5 mg mL⁻¹ for rutin and forsythin, respectively. The relative standard deviations of peak current and migration time for 8 consecutive injections of the standard solution containing 0.1 mg mL⁻¹ each compound were 4.78%, 3.63% and 6.40%, 2.95% for rutin and forsythin, respectively. In addition, levels of the two compounds in traditional Chinese herbal drugs were easily determined.     

Method development and validation for melamine and its derivatives in rice concentrates by liquid chromatography. Application to animal feed samples

An isocratic LC method for the determination of melamine and its degradation products (ammelide, ammeline, and cyanuric acid), used to increase the apparent protein content of rice protein concentrate, has been developed. Method development involved optimization of different RP columns, aqueous mobile phases, pH, phosphate concentration, and temperature. The optimum separation of these compounds was achieved using a Luna CN column (30 °C), 5 mmol L⁻¹ sodium phosphate (pH 5.0) as mobile phase, 1 mL min⁻¹ flow-rate, UV absorbance-DAD detection at 220 nm, and resorcine as internal standard; this enabled separation of these compounds with baseline resolution (values in the 2.1–10.1 range) in about 8 min. Prior to HPLC, the developed sample preparation procedure consisted in a leaching process using the above mentioned mobile phase. Method validation was carried out in rice protein concentrates in accordance with the European Commission decision 2002/657/EC criteria. For this purpose, eight mandatory performance characteristics for the conventional validation approach were determined: calibration graphs, extraction efficiencies, decision limits, detection capabilities, precision (repeatability and within-laboratory reproducibility), accuracy, selectivity, and robustness. The extraction efficiencies for these compounds were in the range 99–100% and the within-laboratory reproducibility at 1.0, 1.5, and 2.0 detection capabilities concentration levels were smaller than 5, 4, and 3%, respectively. Finally, the proposed method was successfully applied to the analysis of other rice protein concentrates and several animal feed samples.     

Comparison of octadecyl-bonded silica and styrene-divinylbenzene copolymer sorbents for trace enrichment purposes fundamental aspects II. Linear chromatographic behaviour of the extracting supports

Summary Two reversed-phase LC systems were investigated by frontal analysis for the determination of linear chromatographic conditions, as defined according to the isotherm concept. The Partisil ODS-3 bonded silica and the PRP-1 polystyrene-divinylbenzene resin were used as stationary phases together withtrans-2-hexen-al as test solute and methanol-water mixtures as mobile phases. Particular attention was paid to the respective influence of the two main parameters which may cause sorbent overloading, that is, the capacity factor (k′) and the solute concentration in the mobile phase (C_m). Provided that k′C_m≤10⁻² M, linear chromatographic behaviour was observed for both sorbents, the maximum capacities of which were found greater than 1mmolg⁻¹.     

A reversed-phase ion-interaction chromatographic method for in-vitro estimation of the percutaneous absorption of water-soluble UV filters

An analytical method based on ion-interaction chromatography with UV detection for simultaneous in-vitro estimation of the percutaneous absorption of the most used water-soluble UV filters in sunscreen cosmetics is proposed. These UV filters were phenylbenzimidazole sulfonic acid, disodium phenyl dibenzimidazole tetrasulfonate, benzophenone-4, and terephthalylidene dicamphor sulfonic acid. The methodology is based on applying the sunscreen containing the target UV filters to human epidermis in a diffusion cell. Analytes are determined in the receptor solution. To ensure skin integrity, screening of the cells was carried out by analytical determination of a marker. Analytical variables such as percentage ethanol, concentration of ion-pairing agent, pH of the mobile phase, and temperature were studied in order to achieve high resolution of the chromatographic peaks in the lowest possible time of analysis. The conditions selected consisted of a mobile phase composed of 35:65 (v/v) ethanol–ammonium acetate buffer solution (pH 4, containing 50 mmol L⁻¹ tetra-n-butylammonium bromide). The chromatographic determination was carried out with the analytical column at 50 °C. UV detection was carried out at the maximum absorption wavelength for each analyte. The limit of detection (3s _y/x /b) ranged from 16 to 65 ng mL⁻¹, depending on the analyte.     

Determination of the antibiotic zinc bacitracin in animal food by high-performance liquid chromatography with ultraviolet detection

Summary A high-performance liquid chromatographic method with ultraviolet detection has been developed for the analysis of the polypeptide antibiotic zinc bacitracin in adulterated animal feed. Firstly, the process for extraction of the antibiotic from the feed was optimized. This process involved extraction from the feed at pH 2, centrifugation, liquid-liquid extraction, then solidphase extraction. The extract obtained was then dissolved in the mobile phase and injected into the chromatograph. The best analytical results were obtained by use of a C₁₈ column with a mobile phase comprising a 50:50 (%,v/v) mixture of 0.3m phosphate buffer, pH 3, containing 20mm sodium dodecyl sulfate (SDS) and 19:1 (v/v) acetonitrile-methanol. The analytical signal (peak area) was monitored at 254 nm. The calibration function was estimated between 200 and 1000 mg L⁻¹. The proposed method was applied to the analysis of zinc bacitracin present in different fortified animal feed products at levels between 5 and 200 mg kg⁻¹. Recovery rates were between 66 and 85% and the relative standard deviation was below 7%.     

Simultaneous RP-LC Determination of Ketorolac and its Piperazinylalkyl Ester Prodrugs

A simple, rapid and selective RP-HPLC method was developed and validated for the determination of ketorolac and five piperazinylalkyl ester prodrugs. A binary isocratic mobile phase composed of a mixture of 65:35 (v/v) 0.02 M phosphate buffer (pH 5.4) and acetonitrile was used on a C₁₈ column (125 × 4 mm, 5 μm). The injection volume was 25 μL and the detection wavelength was 314 nm and the flow rate was 1.5 mL min⁻¹. The method exhibited excellent linearity with R ² of no less than 0.999 and intra-assay and inter-assay precision that were less than the maximum amount allowed according to Horwitz equation. The accuracy was found to be within the allowed ±15%. The limits of detection for the analytes were between 0.060 and 0.220 μg mL⁻¹ and the limits of quantification were between 0.183 and 0.667 μg mL⁻¹. This method was used successfully for the study of the solubility, stability and partition coefficients of piperazinylalkyl ester prodrugs of ketorolac.     

Analysis of Spectinomycin by HPLC with Evaporative Light-Scattering Detection

A high-performance liquid chromatographic method with evaporative light-scattering detection (ELSD) has been developed for analysis of spectinomycin and related impurities. Separation of spectinomycin from structurally related impurities was achieved on a C₁₈ column. The optimized mobile phase was 25 mmol L⁻¹ ammonium acetate (pH 7.5)-methanol, 90:10 (v/v), at a flow rate of 0.6 mL min⁻¹. The temperature of the drift tube of the ELSD was 95°C and the flow rate of carrier gas was 2.2 L min⁻¹. The accuracy, specificity, precision, linearity, sensitivity, and robustness of the method were validated in accordance with ICH guidelines. In addition to determination of spectinomycin and related impurities, the method is also ideal for determination of the salts spectinomycin hydrochloride and spectinomycin sulfate.     

Analysis of Terbumeton and its Major Metabolites by SPE and DAD-HPLC in Soil Bulk Water

A rapid and inexpensive method for simultaneous quantification of terbumeton (TER), and its major potential metabolites (TED; terbumeton-desethyl, TOH; terbumeton-2-hydroxy and TID; terbumeton-deisopropyl) in soil bulk water (SBW) samples is proposed. The analytical method involves extraction–concentration from SBW samples using a graphitized carbon black (GCB) cartridge followed by their separation–detection by reversed-phase high-performance liquid chromatography analysis using a C₁₈ column and a diode array detector. A mobile phase of acetonitrile−0.005 mol L⁻¹ phosphate buffer (pH 7.0) (35:65, v/v) at a flow rate of 0.8 mL min⁻¹ in isocratic elution mode has been used. After optimization of the extraction and separation conditions, this method can be used for the simultaneous determination of investigated compounds in the range of the international limits of 0.1 μg L⁻¹. For TER the detection limit was 0.009 μg L⁻¹ and it was 0.100, 0.550, and 0.480 μg L⁻¹ for TED, TOH, and TID, respectively. The recoveries of TER, TED, TOH, and TID from SBW samples, measured at three levels of concentration range, were found to be between 48.0 and 102.0%. The intra-day precision measured by relative standard deviation (RSD) was always lower than 9.0%.     

Simultaneous determination of hydrochlorothiazide and losartan potassium in tablets by high-performance low-pressure chromatography using a multi-syringe burette coupled to a monolithic column

This contribution describes use of a separation method based on on-line coupling of a multisyringe flow system with a chromatographic monolithic column for simultaneous determination of hydrochlorothiazide and losartan potassium in tablets. The system comprised a multisyringe module, three low-pressure solenoid valves, a monolithic C₁₈ column (25 mm × 4.6 mm i.d.), and a diode-array detector. The mobile phase was 10 mmol L⁻¹ potassium dihydrogen phosphate (pH 3.1)-acetonitrile-methanol (65:33:2 v/v/v) at a flow rate 0.8 mL min⁻¹. UV detection was carried out at 226 nm. The multi-syringe chromatographic (MSC) method with UV spectrophotometric detection was optimized and validated. Results from validation were very good. The analysis time was about 400 s. The method was found to be applicable to routine analysis of both compounds in tablets. The coupling of the monolithic columns with a multi-syringe flow-injection analysis manifold provides an excellent and inexpensive tool to solve the separation problems without use of HPLC instrumentation.     

1,3,5,7-Tetramethyl-8-(<Emphasis Type="Italic">N</Emphasis>-hydroxysuccinimidyl butyric ester)difluoroboradiaza-<Emphasis Type="Italic">s</Emphasis>-indacene as a new fluorescent labeling reagent for HPLC determination of amino acid neurotransmitters in the cerebral cortex of mice

1,3,5,7-Tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)difluoroboradiaza-s-indacene (TMBB-Su), a new BODIPY-based fluorescent probe, was designed and synthesized for the labeling of amino compounds. It was used as a pre-column derivatizing reagent for determination of amino acid neurotransmitters by high-performance liquid chromatography (HPLC). The fluorescence quantum yield in acetonitrile increased from 0.84 to 0.95 when it reacted with amino acid neurotransmitters. Derivatization of TMBB-Su with seven amino acid neurotransmitters was completed within 30 min at 25 °C in 24.0 mmol L⁻¹ pH 7.8 boric acid buffer. The separation was performed on a C₁₈ column with methanol–water–buffer 55:35:10 (v/v) as mobile phase (buffer: 0.10 mol L⁻¹ H₃Cit–0.10 mol L⁻¹ NaOH). Interference from the other concomitant amino acids was eliminated successfully by means of pH gradient elution. With fluorescence detection at 494 and 504 nm for excitation and emission, respectively, the limits of detection (signal-to-noise ratio = 3) were from 2.1 to 12.0 nmol L⁻¹. The proposed method has been used to determine amino acid neurotransmitters in the cerebral cortex of mice with cerebral ischemia at the convalescence stage with satisfactory recoveries varying from 94.9 to 105.2%.     

Isolation and Simultaneous Determination of Coumarin Compounds in Radix Angelica dahurica

For the first time simple, rapid, and systematic methods have been established for preparative isolation and purification of coumarin compounds in an important traditional Chinese Medicine, Radix Angelica dahurica, and for simultaneous determination of several of the compounds in the medicine. Bergapten, imperatorin, and cnidilin, three of the biologically active coumarin compounds, were isolated from the chloroform-soluble fraction of the ethanol extract of Radix Angelica dahurica. After further purification by open column ODS chromatography the purified components were simultaneously determined, with two other coumarins (osthole and isoimperatorin), by reversed phase high-performance liquid chromatography (RP-HPLC) on a C₁₈ column, with methanol–water, 66:34 (v/v), as mobile phase at a flow rate of 0.8 mL min⁻¹. The compounds were detected by UV absorption at 310 nm. Calibration plots for all the coumarins had correlation coefficients close to unity. Limits of detection (S/N = 3) were <92 ng mL⁻¹ and limits of quantification (S/N = 10) were <259 ng mL⁻¹. Mean recovery of the coumarins was in the range 96.7–101.9% and the intra-day and inter-day precision, as relative standard deviation, was <2.3 and <2.9%, respectively. This simple, sensitive, and reproducible method can be used for quality control of Radix Angelica dahurica.     

Liquid chromatographic analysis of the anticancer alkaloid luotonin A and some new derivatives in human serum samples

The quantitation of the natural cytotoxic and anti‐inflammatory alkaloid luotonin A and five recently synthesized derivatives is described, constituting the first report of a HPLC method for the analysis of these compounds in human serum samples. The conditions for the chromatographic separation were optimized and the method was validated for the analysis of these compounds in biological samples according to international guidelines. An RP‐HPLC method with fluorimetric detection and a C₁₈ stationary phase was applied. Different ACN/water mobile phases were assayed, including 0–4% of a mobile phase modifier such as tetrahydrofuran, dioxane or tert‐butyl methyl ether. Isocratic and gradient elution conditions are compared. The influence of pH on the efficiency and resolution of the separation was also considered. The developed method was applied to the determination of luotonins in pooled human serum samples by gradient elution RP‐HPLC using a simple cleanup procedure. The proposed chromatographic method exhibits satisfactory analytical figures of merit, with LOD from 1.0×10^?10 to 2.0×10^?10 M, intraday and interday precision below 6% except for the concentration level closest to LOD, and good agreement between experimental and theoretical concentrations. Therefore, the developed method is suitable, reliable, rapid, and simple.     

Post-column photochemical reaction detection in liquid chromatography based on photosensitized generation of singlet molecular oxygen: Study of reaction parameters and application to the determination of polychlorinated biphenyls

Summary The parameters effecting the sensitivity and selectivity of a photochemical reaction detection scheme based on the reaction of 3-substituted pyrroles with singlet molecular oxygen (¹O₂) in HPLC are reported. Polychlorinated biphenyls (PCBs) were chosen as model compounds for the detection scheme. Following separation by reverse-phase chromatography, PCBs are excited by a Hg pen-ray lamp in a crocheted PTFE photochemical reactor. PCBs that are efficient¹O₂-sensitizers promote ground state O₂ (³Σ_-3) to an excited state (¹Σ_g+ or¹Δ_g) which rapidly oxidizes 3-substituted pyrroles which are added to the mobile phase. Detection is based on the loss of pyrrole. The reaction is catalytic in nature since one analyte molecule may absorb light many times, producing large amounts of¹O₂. Detection limits for 4,4′-dichlorobiphenyl and Aroclors 1242, 1248 and 1254 were improved by 1–2 orders of magnitude over optimized UV-absorbance detection. Configuration of the photochemical reactor and judicious choice of the wavelength used to follow the loss of pyrrole were determined to be the most important factors in terms of sensitivity of the detection scheme. Comparison of three reagents (i.e. 3-substituted-pyrroles) used for trapping¹O₂ demonstrates the effect of substitution on sensitivity and to some extent selectivity of PCB determination.     

RP-HPLC determination of midecamycin and related impurities

Summary A method has been developed for separation and quantitation of midecamycin A₁ and related impurities by high-performance liquid chromatography with evaporative light-scattering detection (ELSD). Chromatographic conditions included use of a Diamonsil C₁₈ column; the mobile phase was 52:48 acetonitrile −0.2 mol L⁻¹ ammonium formate solution (adjusted to pH 7.3 with triethylamine) at a flow rate of 1 mL min⁻¹. The column temperature was 35°C, the shift tube temperature of the ELSD was 105°C, and the gas flow rate of the ELSD was 3.0 L min⁻¹. The response factors of midecamycins in HPLC-ELSD were the same; the linear equation wasy=599292.44x+2868618.04,r=0.9979, the linear range was 5–80 μg,RSD=0.21–1.54%, and theLOD andLOQ were 0.36 and 1.2 μg, respectively. The method was simple, quick, and precise and could be used to determine midecamycin and its related impurities directly.