Column-switching High-performance Liquid Chromatography Research Articles

Column-switching high-performance liquid chromatography electrospray mass spectrometry coupled with on-line of extraction for the determination of mono- and di-(2-ethylhexyl) phthalate in blood samples.

A novel method based on column-switching high-performance liquid chromatography-electrospray mass spectrometry (LC-MS) coupled with an on-line extraction column containing conjugated avidin has been developed for direct injection analysis of di(2-ethylhexyl) phthalate (DEHP) and its metabolite, mono(2-ethylhexyl) phthalate (MEHP), in blood samples. The sample preparation for on-line extraction involved the mixing of blood samples with internal standards, DEHP-d(4) and MEHP-d(4), in LC glass vials. A linear response was found for column-switching LC-MS when tests were conducted within the validated range of 25 to 1000 ng mL(-1) for DEHP and 5 to 1000 ng mL(-1) for MEHP, with correlation coefficients (r) greater than 0.999. In addition, the recoveries of DEHP and MEHP from human plasma were calculated by using this method with on-line extraction, yielding recoveries of up to 91.2% (RSD<5%). We measured the background levels of DEHP and MEHP in six human plasma samples from healthy volunteers and three fetal bovine serum samples for cell-line culture. DEHP and MEHP were not detected in all human plasma samples (N.D. is <25 ng mL(-1) for DEHP, and N.D. is <5.0 ng mL(-1) for MEHP). In contrast, high DEHP contamination of commercially available fetal bovine serum samples was found by this method.

Hydrolysis of N-Phenylimide Herbicide Flumioxazin and Its Anilic Acid Derivative in Aqueous Solutions

Hydrolysis of the N-phenylimide herbicide flumioxazin was studied at pH 2.5-9.0 and 25±1°C by directly analyzing a buffered aqueous solution with column-switching high performance liquid chromatography. Flumioxazin was hydrolyzed via a base-catalyzed opening of the cyclic imide moiety with half-lives of 4.1 days, 16.1 hr and 17.5 min at pH 5.0, 7.0 and 9.0, respectively. The primary hydrolysis product was the anilic acid derivative which was subsequently degraded to the corresponding aniline and dicarboxylic acid via cleavage of the amide linkage under acidic and neutral conditions, along with a partial recyclization to flumioxazin. Kinetic analysis of the pH dependency of each reaction path showed that the acid-catalyzed cleavage of the amide bond in the anilic acid was an intramolecular reaction where the undissociated form of the carboxyl group participated.

Determination of sparteine in human serum by column-switching HPLC with electrogenerated chemiluminescence detection using tris(2,2'-bipyridine) ruthenium (II)

A sensitive column-switching high-performance liquid chromatography with a tris(2,2'-bipyridine) ruthenium(II) electrogenerated chemiluminescence detection method for the determination of sparteine (SP) in human control serum is described. The procedure is based on the enrichment of SP on column 1, followed by transfer of SP to column 2. Column 1 was a Shim-pack MAYI-ODS (10×4.6 mm, SHIMADZU, Tokyo, Japan). Column 2 was a CAPCELL PACK C18 (150×4.6 mm, SHISEIDO, Tokyo, Japan). The electrogenerated chemiluminescence detector was an ECR COMET-3000KANAGAWA (Comet, Kawasaki, Japan). The mobile phase of column 1 was composed of a 15 mM KH2PO4 buffer at pH 6.0 containing 10 mM octanesulfonate-CH3CN (98:2, v/v) and at a flow rate of 0.5 ml/min. The mobile phase of column 2 was composed of 150 mM KH2PO4 buffer at pH 4.0 containing 10 mM 1-octanesulfonate-CH3CN (73:27, v/v) and at a flow rate of 1.0 ml/min. The reagent solution was 0.3 mM Ru(bpy)32+ in 10 mM H2SO4 and at a flow rate of 0.45 ml/min. The sample in the control serum was directly injected to the HPLC. The linear dynamic range was 125 fmol-2 nmol/ml. The mean recovery for 1 nmol/ml was 98%. The limit of quantitation was 125 fmol/ml and the intermediate precision (CV) was 1.8% (1 nmol/ml, n=5).

Method for determining monohydroxybenzo[ a]pyrene isomers using column-switching high-performance liquid chromatography

A method for determining monohydroxybenzo[ a]pyrene (OHBaP) isomers using column-switching high-performance liquid chromatography with fluorescence detection was developed. Eleven of 12 isomers of OHBaP (all except 6-OHBaP) were separated on an alkylamide-type reversed-phase column and, via column-switching, on a β-cyclodextrin-bonded silica gel column. The detection limits for the OHBaPs were in the range 0.3–8 pg/injection ( S/ N=3). By using this method, 1-, 3-, and 9-OHBaPs were identified as major metabolites of benzo[ a]pyrene in vitro by human recombinant P450 1A1. The method was used to determine OHBaPs in the urine of a nonsmoker subject. After enzymatic hydrolysis of the conjugated metabolites by β-glucuronidase/aryl sulfatase, the analytes were selectively adsorbed on blue rayon (a cellulose-supported copper phthalocyanine) from the urine matrix. Methanol as the eluting solvent from the rayon gave the best recoveries of OHBaPs and 1-hydroxypyrene (1-OHP) in the range of 91–103%, which was superior to that of the solid-phase extraction method. 1-OHP, a well-known biomarker of the exposure to polycyclic aromatic hydrocarbons, was simultaneously analyzed. Intra- and interday accuracy values for the determination of 3-OHBaP in 200 ml of urine were 95.5 and 100.9%, and those for 1-OHP were 96.4 and 103.6%, respectively. The intra- and interday precision values were 3.9 and 2.4% for 3-OHBaP and 2.4 and 3.2% for 1-OHP, respectively. In 11 kinds of isomers, only 3-OHBaP was detected in the human urine. Urinary concentration of 3-OHBaP was quantified at 0.5 ng/g creatinine concentration and the 3-OHBaP/1-OHP ratio was approximately 1/130.

Determination of d-alanine in the rat central nervous system and periphery using column-switching high-performance liquid chromatography

A column-switching chiral HPLC system for the determination of minute amounts of d-Ala in mammalian tissues has been established. d-Ala and its l-enantiomer are purified as a dl mixture on a micro-ODS column after precolumn fluorescence derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole and are introduced to a chiral column to determine each enantiomer. The calibration curve of d-Ala spiked into a rat cerebellum sample is linear from 5 to 5000 fmol with a correlation coefficient of 1.0000. The lower limit of quantitation of d-Ala is 5 fmol ( S/ N=5). Within-day and day-to-day precisions of spiked d-Ala (15 fmol) are 3.9 and 4.8% (R.S.D), respectively. With this system, the anatomical distribution of free d-Ala in the rat central nervous system and periphery has been investigated. Among the 22 examined tissues of the rat, the highest amount of d-Ala has been observed in the anterior pituitary gland ( 86.4±9.9 nmol/g wet tissue), and the second highest amount has been observed in the pancreas ( 29.2±5.0 nmol/g wet tissue). Postnatal and day–night changes in d-Ala amounts in the anterior pituitary gland have also been studied. The amount of d-Ala is highest at 6 weeks of age and significantly decreases with age, and the amount of d-Ala is significantly higher during the daytime than during the nighttime.

Automated column-switching high-performance liquid chromatography system for quantifying N-methyl- d- and - l-aspartate

The occurrence and biological significance of the d-amino acids, N-methyl- d-aspartate (NMDA) and N-methyl- l-aspartate (NMLA), have been recently studied in a variety of living organisms. In this study, we established a highly sensitive and reliable fluorometric HPLC system for determining levels of N-methyl-aspartate (NMA). The system comprises fluorescent derivatization of NMA with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and two chromatographic steps: one that separates NMA from other primary amino acids in reverse-phase mode and another that enantioseparates NMDA and NMLA in a normal-phase mode. These two steps are linked by an automated column-switching system. A simple pretreatment step with o-phthalaldehyde to remove primary amino acids that can interfere with sensitivity is also described. The detection limit for NMDA is as low as 5 fmol and the correlation between peak heights and concentrations between 5 fmol and 1 pmol is satisfactory ( r=0.999). Following sample preparation and separation using the column-switching HPLC system, more than 80% of NMDA was recovered from rat liver homogenates spiked with NMDA. This method was employed to determine the levels of NMDA in tissues from bivalves and the results obtained were consistent with the values reported previously.

Determination of bisphenol A in rat brain by microdialysis and column switching high-performance liquid chromatography with fluorescence detection.

A sensitive column switching HPLC-fluorescence detection for determination of bisphenol A (BPA) in rat brain by coupling with microdialysis was developed. A microdialysis probe was inserted into the hypothalamus of rat brain and an artificial cerebrospinal fluid was used for perfusion. BPA in brain dialysate was subjected to a fluorescent derivatization with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl), and the excess reagent was removed by a column-switching technique. Separation was carried out on two ODS semimicro-columns with the mobile phase of acetonitrile-H(2)O-methanol-tetrahydrofuran (55:10:35:2.5, v/v) and acetonitrile-0.1 M acetate buffer (pH 3.0)-methanol (35:10:55, v/v) at a flow rate of 0.10 and 0.15 mL/min for a precolumn and a separation column, respectively. Fluorescence intensity was monitored at 475 nm with excitation of 350 nm. BPA could be sensitively detected at 0.3 ppb in 60 micro L brain microdialysate at a signal-to-noise ratio of 3. By the proposed method, concentrations of BPA in rat brain and plasma were monitored for 8 h after single i.v. or oral administration. It is proved that BPA is capable of penetrating the blood-brain barrier. The ratio of the area under the concentration-time curve of BPA in rat brain to that in blood was estimated to be about 3.0-3.8%.

Determination of fluoxetine enantiomers in rat plasma by pre-column fluorescence derivatization and column-switching high-performance liquid chromatography.

A column-switching HPLC method employing both octadecylsilica (ODS) and chiral columns with fluorescence detection was developed for the determination of enantiomer of fluoxetine (FLX), an antidepressant drug, in rat plasma. Racemic FLX was derivatized with a fluorescent reagent, 4-(N-chloroformylmethyl-N-methyl)amino-7-nitro-2,1,3-benzoxadiazole (NBD-COCl) or 4-(N-chloroformylmethyl-N-methyl)amino-7-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole (DBD-COCl) and the enantiomeric separation of the resultant derivatives was examined on an amylose-based chiral column (CHIRALPAK AD-RH) in the reversed-phase mode. The derivative with NBD-COCl (NBD-FLX) showed a sufficient separation factor (a) and resolution (Rs) compared with that with DBD-COCl. Thus, FLX was derivatized with NBD-COCl and the resultant NBD-FLX was first quantified on the ODS column and then introduced to the CHIRALPAK AD-RH column via a six-port switching valve to examine the enantiomeric ratio. The intra- and inter-day accuracy (97.6-112.7%) and precision (1.47-10.60%) were satisfactory in the range 10-1000 nM FLX and the limit of quantification was approximately 10 nM. The absolute recoveries of FLX with hexane from rat plasma were in the range 87.5-92.2% (n = 3). The method was applied to determine FLX enantiomers in the plasma of rats administered FLX orally, and it was shown that the R-isomer was eliminated faster than the S-isomer.

Relationship Between Urinary Levels of 1-Hydroxypyrene and 3-Hydroxybenzo[ a ]pyrene for Workers Exposed to Polycyclic Aromatic Hydrocarbons

Biomonitoring of workers was carried out in three working areas--an artificial target factory, an aluminum plant, and an electrometallurgy plant--to assess exposure to PAHs. All the 48 hr-voided urine samples of the exposed workers were collected and the 3-hydroxybenzo[ a ]pyrene (3-OHB a P) and 1-hydroxypyrene (1-OHPy) analyzed according to procedures using automated column-switching high-performance liquid chromatography. The exposure profiles indicate an important lag between the excretion of the two metabolites: the maximum of 3-OHB a P urinary excretion is observed 10 to 17 hr after the 1-OHPy maximum, with a much closer correlation ( r = 0.81) than that obtained with both metabolites in real time ( r = 0.21). This delayed excretion agrees with data from animal intoxication studies (intravenous administration or inhalation). Mean ratios of 1-OHPy to 3-OHB a P were studied without lag (varying from 2,230 to 15,330) and with a lag of 16 hr (varying from 940 to 8,390).

Relationship Between Urinary Levels of 1-Hydroxypyrene and 3-Hydroxybenzo[ a ]pyrene for Workers Exposed to Polycyclic Aromatic Hydrocarbons

Biomonitoring of workers was carried out in three working areas--an artificial target factory, an aluminum plant, and an electrometallurgy plant--to assess exposure to PAHs. All the 48 hr-voided urine samples of the exposed workers were collected and the 3-hydroxybenzo[ a ]pyrene (3-OHB a P) and 1-hydroxypyrene (1-OHPy) analyzed according to procedures using automated column-switching high-performance liquid chromatography. The exposure profiles indicate an important lag between the excretion of the two metabolites: the maximum of 3-OHB a P urinary excretion is observed 10 to 17 hr after the 1-OHPy maximum, with a much closer correlation ( r = 0.81) than that obtained with both metabolites in real time ( r = 0.21). This delayed excretion agrees with data from animal intoxication studies (intravenous administration or inhalation). Mean ratios of 1-OHPy to 3-OHB a P were studied without lag (varying from 2,230 to 15,330) and with a lag of 16 hr (varying from 940 to 8,390).

Sequential Analysis of Malic Acid and Both Enantiomers of Lactic Acid in Wine Using a High-Performance Liquid Chromatographic Column-Switching Procedure

A liquid chromatographic column-switching method for the sequential determination of malic acid and both enantiomers of lactic acid in wine is described. The procedure involves the heart cutting of lactic acid enantiomers from a reversed-phase high-performance liquid chromatography chromatogram, retaining them, and back-flushing them through a chiral ligand-exchange column in which they are separated. The method is used to determine the concentration of lactic acid enantiomers in commercial wines. The results are in satisfactory agreement with those of other methods. The malic acid contents of various wines are also determined. The total analysis time for one experiment is approximately 10 min.

Simultaneous determination of olanzapine, clozapine and demethylated metabolites in serum by on-line column-switching high-performance liquid chromatography

An automated method for simultaneous routine quantification of the antipsychotic drugs clozapine, olanzapine and their demethylated metabolites is described. The method included adsorption on a cyanopropyl (CPS) coated clean-up column (10 μm; 10×2.0 mm I.D.), washing off interfering serum constituents to waste, and separation on C18 ODS Hypersil reversed phase material (5 μm; 250×4.6 mm I.D.) using acetonitrile–water–tetramethylethylenediamine (37:62.6:0.4, v/v/v) adjusted to pH 6.5 with concentrated acetic acid. UV-detection was performed at 254 nm. The limit of quantification was 10–20 ng/ml. Relative day to day standard variations ranged between 4.5 and 13.5%. The method is suitable for routine monitoring of olanzapine and clozapine including their demethylated metabolites.

High-performance liquid chromatography determination of flunitrazepam and its metabolites in plasma by use of column-switching technique: comparison of two extraction columns

A study, using on-line column-switching high-performance liquid chromatography, evaluated two different extraction columns for the determination of flunitrazepam and its major metabolites: 7-aminoflunitrazepam, 7-acetamidoflunitrazepam and desmethylflunitrazepam. The procedure was based on the enrichment of benzodiazepines on the extraction column, followed by transfer of the compounds to the analytical column. The two extraction columns were compared: the first column was a BioTrap 500 MS (hydrophobic polymer), 20×4 mm I.D., and the second was a LiChrospher RP-18 ADS, 25×4 mm I.D. The analytical column used was a LiChrospher select B RP-8, 125×3 mm I.D. with 5 μm particle size. The extraction conditions for the two pre-concentration columns, such as extraction temperature, buffer concentration, buffer pH, acetonitrile percentage and flow-rate, were studied for the extraction from plasma of flunitrazepam and its metabolites mentioned above. The mobile phase of the analytical column was isocratic and composed of acetonitrile–20 m M phosphate buffer at pH 2.1 (35:65, v/v) and at a flow-rate of 0.3 ml/min.

Determination of aloesin in rat plasma using a column-switching high-performance liquid chromatographic assay

A column-switching high-performance liquid chromatography (HPLC) method for the determination of aloesin in rat plasma using column-switching and ultraviolet (UV) absorbance detection is described. Plasma was directly injected onto the HPLC system consisting of a clean-up column, a concentrating column, and an analytical column, which were connected with a six-port switching valve. The determination of aloesin was accurate and repeatable, with a limit of quantitation of 10 ng/ml in plasma. The standard calibration curve for aloesin was linear ( r=0.998) over the concentration range of 10–1000 ng/ml in rat plasma. The intra- and inter-day assay variabilities of aloesin ranged from 1.0 to 4.7% and 1.1 to 8.8%, respectively. This highly sensitive and simple method has been successfully applied to a pharmacokinetic study after oral administration of aloesin to rats.

A fluorimetric, column-switching HPLC and its application to an elimination study of LLU-alpha enantiomers in rat plasma.

A method for the enantiomeric determination of 2,7,8-trimethyl-2-(beta-carboxyethyl)-6-hydroxy chroman (LLU-alpha, gamma-CEHC) in rat plasma was developed using high-performance liquid chromatography (HPLC) with a fluorimetric derivatization with 4-N, N-dimethylaminosulfonyl-7-piperazino-2,1,3-benzoxadiazole (DBD-PZ) followed by O-acetylation with acetyl chloride. The proposed HPLC system used two non-chiral columns (phenyl and octadecylsilica) and a chiral column (a modified cellulose type), which were connected via two column-switching valves. A derivatized sample prepared from rat plasma was first separated on the phenyl column, and the fraction including LLU-alpha derivative was introduced to the octadecylsilica column to quantify the concentration of the mixture of S- and R-LLU-alpha. Finally, the LLU-alpha derivative was directly injected into the chiral column to obtain the ratio of the enantiomers. The proposed HPLC system was applied to the enantiomeric determination of LLU-alpha in plasma after intravenous administration of racemic LLU-alpha. S-LLU-alpha was eliminated faster than R-LLU-alpha, and its concentration in plasma decreased to one-third at 2 min after dosing.

Trace determination of urinary 3-hydroxybenzo[ a]pyrene by automated column-switching high-performance liquid chromatography

3-Hydroxybenzo[ a]pyrene (3-OHB[ a]P), one of the metabolites of benzo[ a]pyrene (B[ a]P), has been determined in human urine using an automated column-switching procedure. The hydrolysed biological sample is centrifuged just prior to being injected into a reusable precolumn loop, which is packed with a preparative phase and coupled on-line to a liquid chromatographic (LC) system. A rapid pre-treatment of the hydrolysed sample, consisting of a concentration and a crude clean-up, is performed on the precolumn. The analytes are then non-selectively desorbed with the LC eluent and the sample is cleaned again in three successive purification columns using the direct transfer or “heart-cut” technique. The pre-treatment does not exceed 3 min. and the entire analytical purification and separation procedure takes less than 30 min. Average 3-OHB[ a]P recovery reaches 95% in the 1–50 ng/l range of urine, and the detection limit is 0.1 ng/l urine for a 3 ml injection of hydrolysed urine. The developed method was compared with a more time-consuming off-line method to analyse urines of B[ a]P gavaged rats; the statistical treatment indicates that both methods are in agreement. The method was applied to purify and concentrate the urine samples of workers exposed and apparently unexposed to polycyclic aromatic hydrocarbons (PAHs).

Determination of minute amounts of d-leucine in various brain regions of rat and mouse using column-switching high-performance liquid chromatography

A highly sensitive method for the determination of minute amounts of d-Leu in biological samples was developed. For accurate and sensitive determination, a column-switching system using a micro ODS column and a chiral column was adopted. After pre-column derivatization of d- and l-Leu with NBD-F, the derivatives of the enantiomers were purified on a micro ODS column as a dl mixture. The eluted dl-Leu was then introduced to the chiral column, and each enantiomer was determined. The calibration curve for d-Leu, which was constructed by adding known amounts of d-Leu to a rat hippocampus, was linear from 1 to 1000 fmol ( r>0.999), and the detection limit of added d-Leu was 1 fmol ( S/ N=5). Within-day and day-to-day precisions of d-Leu determination using the same homogenate of rat hippocampus were 5.11 and 5.25% (RSD), respectively. The content of d-Leu in rat hippocampus was 0.69 nmol/g wet tissue (the percentage of d-enantiomer for total Leu was 0.97%), which was consistent with the reported value. The distribution of d-Leu in mouse brain was also investigated, and the presence of d-Leu in various regions of the mammalian brain was first observed.

Determination of vitamin D 2 in emulsified nutritional supplements by solid-phase extraction and column-switching high-performance liquid chromatography with UV detection

This paper deals with a method for solid-phase extraction of trace amounts of vitamin D 2 (VD 2, 19 ng/g) from emulsified nutritional supplements, which contain 50 kinds of compounds, followed by column-switching high-performance liquid chromatography (HPLC) with UV detection at 265 nm. VD 2 is present at 1000–20 000 000 times lower concentration than other components. Bond Elut C 18 cartridge was chosen as for the emulsified nutritional supplements after comparison with eight other types. A sample solution was applied to the solid-phase extraction cartridge and VD 2 was eluted by methanol followed by HPLC. The effects of sample pH, eluent composition and eluate volume on the retention and elution of VD 2 on Bond Elut C 18 cartridge were examined. The resulting method was simple, rapid (analysis time: ∼20 min), sensitive (detection limit: ∼0.1 ng per injection (200 μl) at a signal-to-noise ratio 3:1), and reproducible (relative standard deviation: ∼6.2%, n=5). The calibration graph for VD 2 was linear in the range of 0.1–3 ng per injection (200 μl). Recovery of VD 2 was ∼80% by the standard addition method.

Quantitative Analysis of Niacin in Food by Column-Switching High Performance Liquid Chromatography

Quantitative Analysis of Niacin in Food by Column-Switching High Performance Liquid Chromatography

Direct Determination of Platelet-Produced Thromboxane B2 in Human Serum by Column-Switching High-Performance Liquid Chromatography with Fluorescence Detection

A high-performance liquid chromatographic method for the direct determination of platelet-produced thromboxane B2 in human serum is described. The method is based on direct derivatization of the thromboxane B2 in human serum with 6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone-3-propionyl carboxylic acid hydrazide. The derivatization reaction proceeds in aqueous solution (or serum sample) in the presence of pyridine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at 37˚C. The resulting fluorescent derivative of thromboxane B2 is separated by column-switching chromatography using a first column (YMC-Pack C4) for the trap and clean-up of the derivative and a second column (YMC-Pack Ph) for complete separation of the derivative. The derivative is detected spectrofluorometrically at 445 nm with excitation at 367 nm. The detection limit (signal to noise ratio=3) for thromboxane B2 is 10 pmol (3.7 ng)/ml serum. The present method is highly sensitive and simple without any clean-up, such as in conventional liquid-liquid and solid-phase extraction.