Determination of metoprolol in human plasma by column switching high-performance liquid chromatography

Summary Retention characteristics of metoprolol have been studied in reversed phase mode on RP2, RP8 and CN columns. The plots of retention time as a function of the acetonitrile content and of the ionic strength of the mobile phase permitted the choice of the best conditions to separate metoprolol from plasma components by switching of these three types of columns.Human plasma (0.5–1 ml) diluted with water is first injected on a RP2 column (25–40 m particle diameter, prepared by dry packing) and rinsed with water. The sample is then back eluted with acetonitrile-0.022 M acetate buffer (7525, v:v) and switched to a CN column (10 cm long, 5 m particle diameter). The heart cut of the eluate is selected and loaded on a RP8 analytical column (25 cm long, 5 m particle diameter) with acetonitrile-0.088 M acetate buffer (7525, v:v) as mobile phase.Auto-sampler and switching valves are actuated automatically by a computing integrator based on a fixed time schedule. The duration of one cycle is about 30 min, but the last analytical step is about 15 min and represents the time interval between two injections. Metoprolol, its alpha-hydroxy metabolite and the internal standard are detected by fluorescence (_ex= 225 nm; _em > 320 nm).Presented at the 14th International Symposium on Chromatography London, September, 1982     

Separation of alkylnaphthalenes by preparative liquid chromatography using column switching systems

Summary The paper describes the separation of the mixture of alkynaphthalenes from distillation cuts of a pyrolysis oil, by preparative liquid chromatography on silica. The design of the system permits the connection of the columns to form multicolumn systems.The samples were first separated on a single column. The mixtures were further separated using two-column chromatography systems.The obtained fractions were analyzed by capillary gas chromatography. In most cases a substantial increase in the concentrations of the individual components was achieved. In several cases, pure compounds have been obtained. Separation efficiency increases in the following order: single column, two directly coupled collumns, two-step switching chromatography, heartcutting.     

Bioanalysis of fluorouracil applying liquid chromatography and valve switching

Summary Fluorouracil has been analyzed with a reversed-phase ion-pair liquid chromatographic system applying PRP-1 as the stationary phase. A valve switching technique was applied for the separation of the analyte and some other chemotherapeutic agents. The described procedure gives a rapid screening for the analyte in urine and plasma samples without interferences from late eluting solutes. Biological samples are pretreated by means of a liquid-liquid extraction resulting in a recovery of about 52% for plasma and urine with minimum detectable amounts of about 5ng/ml. The sensitivity and the selectivity of the procedure allows the monitoring of fluorouracil in body fluids and subsequent pharmaco-kinetic studies of the fluoropyrimidine.     

Mixed mode chromatography via column switching for the simultaneous HPLC analysis of lonic and non-lonic nucleic acid constituents

Summary A method is presented for the separation of purine and pyrimidine nucleotides, nucleosides and bases in one analysis. This method uses both the ion-exchange and the reversed-phase modes for the analysis of these compounds. The modes are coupled through automatic switching valves. The use of these valves permits the isolation of either column during the analysis. The nucleotides are separated on an anion-exchange column and the nucleosides and bases on a C-18 column. Total analysis time is approximately one hour, and a very small volume of a sample can be analyzed. The technique is flexible and mobile phase conditions for one column can be altered without affecting the resolution on the other column. The method is applicable to biological matrices.     

Coupled column liquid chromatography for the rapid determination of N-methylcarbamates and some of their main metabolites in water

Summary A sensitive and selective coupled-column liquid chromatography (LC-LC) method was developed for the trace level determination of some N-methylcarbamates and some of their main metabolites as aldicarb, aldicarb-sulphoxide, aldicarb-sulphone, carbofuran and 3-hydroxicarbofuran in drinking and ground waters. The limit of determination can be reduced to 0.1 μg.L⁻¹ by solid phase extraction with a subsequent evaporation step. Environmental samples spiked at 0.1 μg.L⁻¹ were preconcentrated off-line with graphite carbon and then analyzed by LC-LC with UV detection yielding average recoveries between 81–109% (n=5) with RSD between 5–9%. The overall procedure allowed a sample throughput of up to 30 samples per day.     

A column switching technique for the determination of lidocaine and its metabolites in horse urine

Summary A rapid method is described for the determination of lidocaine and its metabolites in horse urine using a column switching technique and HPLC analysis. This procedure offers a sensitive assay without the need for time consuming extractions.     

Sensitive biomonitoring of phthalate metabolites in human urine using packed capillary column switching liquid chromatography coupled to electrospray ionization ion-trap mass spectrometry

A rapid and sensitive method for the determination of the phthalate monoesters monoethyl phthalate (MEP), monobutyl phthalate (MBP), monobenzyl phthalate (MBzP) and monoethylhexyl phthalate (MEHP), in human urine, using packed capillary column liquid chromatography coupled to electrospray quadrupole-ion trap mass spectrometry (ESI-QITMSⁿ) has been developed. Sample volumes of 200 L of deconjugated and diluted urine were loaded onto a precolumn of 30 mm×0.32 mm I.D. packed with Hypercarb 5 m particles, using a sample carrier consisting of acetonitrile/water (15/85, v/v, adjusted to pH 2 using HCl) with a flow rate of 20 L/min. Backflushed elution onto a 100 mm×0.32 mm I.D. analytical column packed with 5 m Hypercarb particles was conducted using a tetrahydrofuran/water gradient where both solvents contained 10 mM ammonium acetate, at a flow rate of 4 L/min. Determination of the monophthalates was achieved within 8 min. Ionization was performed in the negative mode and the analytes were observed as [M-H]^– at m/z=193.1, 221.1, 255.1 and 277.0 for MEP, MBP, MBzP and MEHP, respectively. Quantification was performed in the multiple reaction monitoring (MRM) mode monitoring the fragments at m/z=121.1, 177.0, 183.0 and 233.0 for MEP, MBP, MBzP and MEHP, respectively. The method was validated over the concentration range 2.5–125 ng/mL in pretreated urine samples, corresponding to 25–1250 ng/mL untreated urine, yielding correlation coefficients in the range 0.996–0.999. The within-assay (n=6) and between-assay (n=6) repeatabilities were in the range 4.0–18% and 4.8–15% RSD, respectively. The mass limits of detection were in the range 32–70 pg, corresponding to concentration limits of detection of 1.6–3.5 ng/mL of untreated urine.     

Supported liquid membrane work-up in combination with liquid chromatography and electrochemical detection for the determination of chlorinated phenols in natural water samples

Summary A sample preparation method has been developed for the determination of chlorinated phenols in water. The method is based on a supported liquid membrane extraction system connected on-line to liquid chromatography with electrochemical detection. The supported liquid membrane technique utilizes a porous PTFE membrane. The membrane is impregnated with an organic solvent which forms a barrier between two aqueous phases and enables selective extraction. The technique can easily be coupled in a flow system. In this investigation five chlorinated phenols (1–5 chlorine atoms) were extracted from natural water samples. Extraction for 30 minutes resulted in detection limits of approximately 25 ng L^–1.     

Use of two-dimensional liquid chromatography in the analysis of additives in cellulose acetate polymer

Summary The capability of elution-elution multi-dimensional liquid chromatography was investigated. A column scaling approach was evaluated for the quantification of low-molecular-weight additives in cellulose acetate. A small-bore (1-mm i.d.) gel-permeation column was used to separate the higher-molecular-weight polymer from the lower-molecular-weight components. Once separated these additives were transferred to a C₁₈ reversed-phase column via a switching valve. The reversed-phase system successfully separated and quantified individual additives.Analysis time for an ultraviolet inhibitor, Tinuvin^®P, in cellulose acetate, including re-equilibration, was approximately 30 minutes. Both accuracy and precision were good. Precision over a three day period was about 1.5%.     

Determination of D- and L-amino acids in biological samples by two-dimensional column liquid chromatography

Summary A two-dimensional, column liquid chromatographic system is used for the determination of the D- and L-enantiomers of amino acids in biological samples. Separation of the amino acids is first on ion-exchange column by gradient elution with a sodium citrate-sodium chloride buffer. Enantioseparation is by subsequent injection of 3 μl heart-cuts of the individual amino acids onto a second column with a chiral crown ether stationary phase. Finally, fluorescence detection is after post-column labelling of the amino acids using ano-phthalaldehyde-2-mercaptoethanol reagent solution. The high separation power and selectivity of the system allow processing of complex samples with hardly any additional treatment and the determination of small quantities of D-amino acids in the presence of excess L-form. Applicability of the system is illustrated by the determination of D- and L-aspartate, serine, glutamate and alanine in various complex biological samples, such as protein hydrolysates, urine and biotechnological and food samples. Data are given on detectability, repeatability and linearity.     

Determination of D- and L-amino acids in biological samples by two-dimensional column liquid chromatography

Summary A two-dimensional, column liquid chromatographic system is used for the determination of the D- and L-enantiomers of amino acids in biological samples. Separation of the amino acids is first on ion-exchange column by gradient elution with a sodium citratesodium chloride buffer. Enantioseparation is by subsequent injection of 3 l heart-cuts of the individual amino acids onto a second column with a chiral crown ether stationary phase. Finally, fluorescence detection is after post-column labelling of the amino acids using ano-phthalaldehyde-2-mercaptoethanol reagent solution. The high separation power and selectivity of the system allow processing of complex samples with hardly any additional treatment and the determination of small quantities of D-amino acids in the presence of excess L-form. Applicability of the system is illustrated by the determination of D- and L-aspartate, serine, glutamate and alanine in various complex biological samples, such as protein hydrolysates, urine and biotechnological and food samples. Data are given on detectability, repeatability and linearity.     

Determination of acyclovir in dog plasma by HPLC using a column switching technique

Summary A new HPLC-UV method has been developed and validated for the pharmacokinetic linearity study of Telviran^? tablets containing 200, 400, and 800 mg acyclovir. RP-18 solid phase extraction has been developed for sample preparation. Guanosine (9-[β-D-ribofuranosyl]-guanine) was used as internal standard. The separation was carried out on an ODS Hypersil (5 μm, 200×4.5 mm) analytical column, supplied with a 20 mm guard column containing the same packing material. A column switching technique was applied for the elimination of the endogenous compounds eluting with longer retention times than the investigated compounds, so the analysis time was considerably shorter compared with the time of gradient elution. The eluent was 0.5% triethylamine in water, the pH was adjusted with orthophosphoric acid (85%) to pH5. The detection was performed at 254 nm. The calibration curve was linear in the concentration range 10–5000 ng mL⁻¹. The new bioanalytical method was successfully applied for a pharmacokinetic linearity study in dogs. Presented at Balaton Symposium on High Performance Separation Methods, Siófok, Hungary, September 1–3, 1999     

Application of a polysiloxane-based extraction method combined with column liquid chromatography to determine polycyclic aromatic hydrocarbons in environmental samples

Silicone rods with a diameter of 1 mm and 10 mm long were used to extract polycyclic aromatic hydrocarbons (PAHs) from water samples and for the rapid screening of highly contaminated waste material. The rods were placed in a 15 ml glass vial for the extraction of the analytes, which involved shaking (300 min⁻¹) the sample for 3 h. After extraction the rods were placed into 250 μl inserts of 2 ml vials filled with 100 μl of an acetonitrile-water mixture (4:1) and desorption was performed with sonication for 10 min. Then the PAHs were determined using LC and fluorescence detection. Recoveries of the rod extraction ranged between 62 and 97% and the detection limits were between 0.1 and 1.2 ng l⁻¹. These results are comparable with those of stir bar sorptive extraction (SBSE). Although the rods are reusable, their low price means they can be discarded if contaminated, eliminating the need for expensive cleaning. One disadvantage compared to SBSE is the longer extraction time needed to reach equilibrium.     

Development and validation of a liquid chromatography method for analysis of colistin sulphate

Summary The peptide antibiotic Colistin has been successfully separated into more than thirty components on a YMC-Pack Pro, C-18 column with a mobile phase of acetonitrile—sodium sulphate (0.7% m/v)—phosphoric acid (6.8% v/v dilution of 85% m/m H₃PO₄)—water (21.5∶50∶5∶23.5) at 1.0 mL per min and detection at 215 nm. Baseline resolution of the major components, colistin A and B, and many minor components was obtained. Robustness was evaluated by performing a full-factorial design experiment. The method showed good selectivity, repeatability, linearity and sensitivity.     

Comparison of provitamin A determination by normal-phase gravity-flow column chromatography and reversed-phase high performance liquid chromatography

Summary The provitamin A content of some food samples was determined by methods involving MgO: Hyflosupercel gravityflow column chromatography (GFCC) and reversed phase high performance liquid chromatography (HPLC), the quantitation being done by external standardization (HPLC-ES) or internal standardization (HPLC-IS) with Sudan. The results obtained with - and -carotene in carrots, -carotene and -cryptoxanthin in papaya and -carotene in tomato and kale agreed well, showing that any of the these techniques can be used, provided the analysis is done under optimum conditions. Good separation of the different provitamins using GFCC depends on the analyst's skill and visual acuity. HPLC-ES required a constant supply of provitamin standards, thus the varying purity of commercially available standards and the high instability of these compounds could pose grave problems. Due to the stability of Sudan, HPLC-IS appeared to be the method of choice although passage of the extract through a MgO: Hyflosupercel minicolumn was required prior to injection to separate chlorophylls, dihydroxy- and polyoxycarotenoids which would otherwise elute with Sudan. Nonconformity of the Sudan structure to those of the provitamins did not effect the quantitative results. The chromatographic separation, identity and quantification of the provitamins could be more easily established by using HPLC-IS, complemented with GFCC.     

An integrated coupled column liquid chromatography system for the determination of leukotriene metabolites in biological samples

Summary A fully integrated chromatographic system was developed for the determination of leukotrienes in biological samples using photodiode-array detection (PDAD), which eliminates time consuming manual sample handling steps. A special solid phase extraction, (SPE) methodology for leucotriene metabolite stability was developed which increased the recoveries and eliminates the contamination risk of biological samples. The inherent instability (autooxidation) of many of the leukotriene mediators, and the adsorption effects onto exposed surfaces in vials and in the chromatographic system were found to be very important parameters to control in order to circumvent high loss of sample analytes. By binding the cell supernatants to the functionalities of the SPE support stabilised these mediators. Cell culture samples were eluted through a disposable C₁₈ SPE column. The SPE columns were allowed to thaw and deposited in an automated sample handling unit (ASPEC XL). Desorption of the analytes was followed by a second on-line SPE step, to eliminate remaining interfering matrix compounds. Typical recoveries when stored at −70°C were in-between 55–97% except for (LTE₄) which was found to be around 40% after 72 days of storage. Seven reversed-phase packings were studied. Selectivity factors, as well as the separation efficiencies, were found to differ for the various C₁₈ bonded silica stationary phases. This integrated on-line column liquid chromatographic system was applied to the determination of leukotriene B₄, leukotriene C₄, leukotriene D₄, leukotriene and E₄ in human cell extracts using prostaglandin B₂ as the internal standard. More than 1500 biological samples were analysed. Some validation data are presented for unattended operations.     

Hollow fiber supported ionic liquid membrane microextraction for determination of sulfonamides in environmental water samples by high-performance liquid chromatography

By using ionic liquid as membrane liquid and tri-n-octylphosphine oxide (TOPO) as additive, hollow fiber supported liquid phase microextraction (HF-LPME) was developed for the determination of five sulfonamides in environmental water samples by high-performance liquid chromatography with ultraviolet detection The extraction solvent and the parameters affecting the extraction enrichment factor such as the type and amount of carrier, pH and volume ratio of donor phase and acceptor phase, extraction time, salt-out effect and matrix effect were optimized. Under the optimal extraction conditions (organic liquid membrane phase: [C₈MIM][PF₆] with 14% TOPO (w/v); donor phase: 4 mL, pH 4.5 KH₂PO₄ with 2 M Na₂SO₄; acceptor phase: 25 μL, pH 13 NaOH; extraction time: 8 h), low detection limits (0.1–0.4 μg/L, RSD ≤ 5%) and good linear range (1–2000 ng/mL, R² ≥ 0.999) were obtained for all the analytes. The presence of humic acid (0–25 mg/L dissolved organic carbon) and bovine serum albumin (0–100 μg/mL) had no significant effect on the extraction efficiency. Good spike recoveries over the range of 82.2–103.2% were obtained when applying the proposed method on five real environmental water samples. These results indicated that this present method was very sensitive and reliable with good repeatabilities and excellent clean-up in water samples. The proposed method confirmed hollow fiber supported ionic liquid membrane based LPME to be robust to monitoring trace levels of sulfadiazine, sulfamerazine, sulfamethazine, sulfadimethoxine and sulfamethoxazole in aqueous samples.     

A rapid method for the determination of methoprene in tobacco by high performance liquid chromatography

Summary A rapid sample preparation procedure combined with a short reversed-phase HPLC separation for the quantitation of methoprene residue in tobacco samples is described. A ground tobacco sample of 0.5 g is mixed with 3 mL of 2-propanol. The mixture is extracted for twelve minutes with the aid of sonication at an elevated temperature (45–55°C) and then filtered through a 0.45 m disposable filter prior to injection on HPLC. No sample cleanup or solvent evaporation step is required. Chromatographic analysis is performed on a C-18 column and the analysis time is 12.5 minutes. The detection limit for methoprene in tobacco samples is one part per million (g/g).     

Residue analysis by multicolumn liquid chromatography — Herbicides in cereals

Summary The application of multicolumn HPLC in the determination of residues of a new herbicide and its main metabolite in green plants, grains and straw of different cereals is demonstrated. The columns are combined on-line by column switching and UV-detection is used. Due to the high separation power of multidimensional liquid chromatography only a simple and fast sample pretreatment is required resulting in a detection limit of 10^–8 g/g.