On-line Column-switching System Research Articles

Fast liquid chromatography-mass spectrometry reveals side chain oxysterol heterogeneity in breast cancer tumour samples

Oxysterols can contribute to proliferation of breast cancer through activation of the Estrogen Receptors, and to metastasis through activation of the Liver X Receptors. Endogenous levels of both esterified and free sidechain-hydroxylated oxysterols were examined in breast cancer tumours from Estrogen Receptor positive and negative breast tumours, using a novel fast liquid chromatography tandem mass spectrometry method. Multiple aliquots of five milligram samples of 22 tumours were analysed for oxysterol content to assess intra- and inter-tumour variation. Derivatization was performed with Girard T reagent (with and without alkaline hydrolysis) and sample clean-up was performed using a robust automatic on-line column switching system (“AFFL”). Oxysterols were separated isocratically on a 2.1 mm inner diameter column packed with ACE SuperPhenylHexyl core shell particles using a mobile phase consisting of 0.1% formic acid in H2O/methanol/acetonitrile (57/10/33, v/v/v) followed by a wash out step (0.1% formic acid in methanol/acetonitrile, 50/50, v/v). The total analysis time, including sample clean-up and column reconditioning, was 8 min (80% time reduction compared to other on-line systems). Analysis revealed large intra-tumour variations of sidechain oxysterols, resulting in no significant differences in endogenous oxysterols levels between Estrogen Receptor positive and Estrogen Receptor negative breast cancers. However, a correlation between esterified and free 27-hydroxycholesterol was observed. The same correlation was not observed for 24S-hydroxycholesterol or 25-hydroxycholesterol. The oxysterol heterogeneity of tumour tissue is a critical factor when assessing the role of these lipids in cancer.

Direct Injection Liquid Chromatographic Technique for Simultaneous Determination of Two Antihistaminic Drugs and Their Main Metabolites in Serum

A very simple liquid chromatographic technique was developed and validated for the simultaneous determination of 2 antihistaminic drugs, loratadine (LT) and terfenadine (TR), and their major active metabolites, desloratadine (DL) and fexofenadine (FX), respectively, in human serum. LT, DL, TR, and FX from directly injected serum samples were enriched on a protein-coated RP8 silica precolumn (10 x 4.6 mm id) while serum constituents, such as proteins and salts, were eluted to waste. Using an online column-switching system, the drugs and their metabolites were quantitatively transferred and separated on a second analytical column (Shim-pack 5 microm particle size cyanopropyl, 250 x 4.6 mm id) followed by ultraviolet detection at 243 nm for LT and DL and 220 nm for TR and FX. Very good precision, accuracy, and linearity were obtained over the range of 10-1000 ng/mL for LT and DL, 10-500 ng/mL for TR, and 10-3000 ng/mL for FX in human serum. High extraction recoveries from serum ranging from 96.03 to 98.19, 95.44 to 97.26, 95.61 to 98.17, and 95.60 to 97.89 for LT, DL, TR, and FX, respectively, were obtained.

Genotoxicity Screening Using Biocatalyst/DNA Films and Capillary LC−MS/MS

Detection of DNA adducts can serve as a basis for genotoxicty screening of new chemicals and drugs. We report here a simple, sensitive procedure for this purpose using films containing DNA and a biocatalyst to mimic the metabolic action of human liver cytochrome P450s. DNA adducts formed from an in-situ-generated toxic metabolite (styrene oxide) were detected at subpicomole levels after neutral thermal hydrolysis of the DNA films and analysis with capillary liquid chromatography with on-line column preconcentration and MS/MS detection. An on-line column switching system allowed for increased sample loading volume and analyte preconcentration. This approach provides an estimate of the relative rate of DNA damage.

Chromatographic assay and pharmacokinetic studies of propofol in human serum.

A high-performance liquid chromatographic system with automated precolumn extraction was developed for the determination of propofol in human serum. Propofol of directly injected serum sample was enriched on a protein-coated mu Bondapak phenyl precolumn while serum constituents such as proteins and salts were eluted to waste. Thereafter, using an on-line column-switching system, the drug was quantitatively transferred and separated on a second analytical column followed by spectrophotometric determination at 270 nm. Good precision, accuracy and linearity were obtained over a range of 30-3000 ng/mL propofol in human serum. The developed method proved to be fast, simple, reproducible, reliable and therefore convenient for propofol monitoring from serum. The recovery of propofol in serum samples from the lowest to the highest concentration ranged from 96.84 to 100.16% (n = 5). The assay was applied to study the pharmacokinetic of the drug in six women undergoing elective caesarean section under general anaesthesia induced with a single intravenous bolus dose of propofol (2.5 mg/kg).

Quantification of trovafloxacin in serum by high-performance liquid chromatography with on-line solid-phase extraction

A rapid, simple, accurate and sensitive liquid chromatographic assay with on-line solid-phase extraction is described for determination of trovafloxacin in human serum. Samples were deproteinized with acetonitrile and injected on to an NH2 extraction column for sample clean-up. Thereafter, an on-line column-switching system was used for quantitative transfer of the drug to a C18 analytical column. Separation was performed by ion-pair chromatography and detection was by ultraviolet absorbance at 275 nm. Recovery was 98.5%. The linear range was from 0.25 to 20μg mL−1, with a correlation coefficient of 0.999. Detection limit was 0.1 μg mL−1 from extraction of 25 μL serum.

On-line solid-phase extraction of cephalosporins

Using a single, on-line solid-phase sample clean-up procedure, serum concentrations of ten cephalosporins (cefaloridine, cefalotin, cefamandole, cefazolin, cefodizime, cefoperazone, cefoxitin, ceftizoxime, ceftriaxone and cefuroxime) were assayed on a C 18 column, using UV detection. Serum samples diluted with water were directly injected, without any sample preparation, into the extraction column (alkylamino packing) where the drugs were separated from the serum components. Using an on-line column-switching system, cephalosporins were transferred and separated on the analytical column by ion-pair high-performance liquid chromatography. The proposed method was rapid and efficient: it requires a small serum volume (10 μl) and it can be automated. Recovery was nearly complete. Detection limits varied from 0.5 to 2 μg/ml from extraction of 10 μl of serum. The method was highly specific even in the presence of other drugs commonly found in clinical samples and provides a rapid, simple technique suitable for use in routine microbiological practice.

Determination of meropenem in serum by high-performance liquid chromatography with column switching

A rapid, accurate and sensitive liquid chromatographic assay with on-line solid-phase extraction for determination of meropenem in serum is described. Sample was directly injected onto the extraction column for sample clean-up and extraction. Thereafter, using an on-line column-switching system the drug was quantitatively transferred and separated on a C 18 analytical column. Ultraviolet absorption at 298 nm was used for detection. The assay was linear from 1 to 100 μg/ml. Recovery was 98.5%. Based on a 20-μl sample volume (serum–water, 1:1, v/v), detection limit was 0.1 μg/ml. An application of the method to study the pharmacokinetics of meropenem is given.

Column-Switching High-Performance Liquid Chromatographic Assay for Minocycline of Nude Mice Serum

A rapid, direct, accurate and sensitive liquid chromatographic assay with on-line extraction for minocycline of nude mice serum is described. Serum was directly injected onto the extraction column where the drug was separated from the serum concomitants using a solid phase extraction procedure based on cation exchange. Using an on-line column switching system, minocycline was quantitatively transferred and separated on the analytical column. Ultraviolet absorption at 352 nm was used for detection. The assay was linear from 0.2 to 10 μg/mL with a correlation coefficient of 0.999. Detection limit was 50 ng/mL. Recovery was 97%. The method has been developed for the determination of minocycline levels of nude mice serum collected from a study designed to evaluate the potential for tumor anti-angiogenic effects of this drug.

On-line solid-phase extraction of ceftazidime in serum and determination by high-performance liquid chromatography.

A column-switching high-performance liquid chromatographic assay is described for the determination of ceftazidime (a third-generation cephalosporin) in human serum. The method does not require prior sample pretreatment. Serum is directly injected in a first chromatographic column for sample clean-up and extraction. Thereafter, using an on-line column-switching system, the drug is quantitatively transferred and separated on a second, analytical column followed by determination using ultraviolet absorption at 258 nm. The technique allows direct, rapid, precise, and simple determination of ceftazidime in serum over the range of 1-250 micrograms/ml using 12.5 microliters of serum. This method was applied to study the pharmacokinetics of the drug in patients undergoing vascular surgery.

On-line solid-phase extraction of piroxicam prior to its determination by high-performance liquid chromatography

A direct method for the determination of piroxicam in plasma is described. Plasma is directly injected onto the extraction column (10 mm × 2 mm I.D., packed with 40-μm Bond Elut C 2) where piroxicam is separated from the plasma concomitants using a solid-phase extraction procedure. Using a laboratory-made on-line column-switching system, the drug is quantitatively transferred and separated on the analytical column (15 cm × 4.6 mm I.D., Supelcosil LC18 DB, 5 μm) followed by determination using ultraviolet absorption at 331 nm. Validation of the method demonstrated a good recovery (100%), sensitivity (limit of determination 0.2 μg/ml, based on a 20-μl sample volume), accuracy and precision (better than 5%). The developed method has been adopted for studying the steady-state pharmacokinetics of the drug.