Mark E. Hinsdale

Analysis of nitrate in biological fluids by capillary electrophoresis.

Nitrite and nitrate represent the products of the final pathway of nitric oxide metabolism. These two ions were analyzed by capillary electrophoresis (CE) in serum, cerebrospinal fluid, urine and tissue homogenates by mixing the sample with acetonitrile containing NaBr as an internal standard, followed by centrifugation. The supernatant was injected hydrodynamically on a capillary 50 cm x 75 microns (I.D.) and electrophoresed at 6 kV (reversed polarity) in 1.4% sodium chloride in phosphate buffer for 13 min with detection at 214 nm. In addition to removal of the proteins, acetonitrile caused sample stacking. Urinary nitrate analysis by CE was compared to that by the enzymatic Aspergillus nitrate reductase method, with a correlation coefficient of 0.96.

Effect of pH and ions in the sample on stacking in capillary electrophoresis

Abstract Concentrating the sample on the capillary called “stacking” is a simple technique that overcomes the poor detection limits of capillary electrophoresis (CE). Understanding the factors that affect the stacking mechanism is vital. Two general stacking methods are used in CE: (1) low ionic strength buffer in the sample (LISS) and (2) stacking by inclusion of acetonitrile (AS) in the sample. As the sample volume, especially in the latter method, is greatly increased we show that the pH, buffer type and ionic strength of the sample affect greatly the plate number, resolution and migration time. Surprisingly, we find inclusion of a pH, molarity or ion type in the sample, different from that of the separation buffer, can greatly improve the resolution and the stacking for some components of the sample, especially for the acetonitrile induced stacking.

Analysis of isoflavones by capillary electrophoresis

A simple capillary electrophoresis method is described for the assay of several isoflavones and coumestrol isolated from plant extracts. The method has good reproducibility; it compares well to HPLC, and it can be performed in less than 10 min.

Analysis of the antiepileptic drug keppra by capillary electrophoresis

A simple and rapid method for determination of the new antiepileptic drug keppra (levetiracetam) by capillary electrophoresis in borate buffer containing sodium dodecyl sulfate is described. The serum was injected without any treatment. The method compared well to high performance liquid chromatography. The mean of keppra in the serum of 35 patients was 25 mg/l (range 7-77 mg/l).

Analysis of ibuprofen in serum by capillary electrophoresis

A rapid method for analysis of the analgesic drug ibuprofen in serum by capillary zone electrophoresis in a borate buffer 160 mmol/l pH 8.5 is described. The method involves deproteinization with acetonitrile to remove serum proteins followed by direct injection on the capillary. The recoveries of standards added to the serum were 84-92%. The method is suited for analysis of samples with concentrations > 10 mg/l. Many other analgesics such as ketoprofen, daypro and salicylates can also be determined by this method.

Xanthine analysis in biological fluids by capillary electrophoresis

Xanthine, a precursor of uric acid, is measured here in serum, urine, and cerebrospinal fluids by capillary electrophoresis (CE) after deproteinization with acetonitrile. The migration time is about 7.5 min with a minimum detection limit of 0.4 mg/l. Different purines and pyrimidines did not interfere with the determination. The method demonstrates the suitability of the CE for determination of small molecules present in a complex matrix at levels of ca. 1mg/l. It also demonstrates that acetonitrile deproteinization is a simple and effective method for preparing samples for CE, allowing a large volume to be introduced into the capillary.

Analysis of glutathione by capillary electrophoresis based on sample stacking

Glutathione is a small peptide, which participates in cellular oxidation‐reduction and detoxification. It is present in most biological tissues at different concentrations. The oxidized and reduced forms of the peptide were measured in erythrocytes and myocardial tissue by capillary electrophoresis based on stacking. After tissue homogenization or hemolysis of the red blood cells, the samples were deproteinized with acetonitrile and injected filling about 13% of the capillary volume. The electrophoresis was performed at 10 kV using a separation buffer of 250 mM borate, 50 mM Tris, pH 8.0. Sample stacking increased the sensitivity of detection by 10–20‐fold.

Analysis of the contrast agent iopamidol in serum by capillary electrophoresis

A rapid method for the analysis of the contrast agent iopamidol (isovue)in serum by CE is described. The method involves deproteinzation with acetonitrile to remove serum proteins followed by direct capillary zone electrophoresis. It is rapid(about 10 min) and sensitive. Other contrast agents and compounds used for renal function tests such as iohexol, iothalamic acid and p-aminohippuric acid can be detected by the same method. The use of acetonitrile in this method to remove serum proteins results in sample stacking allowing the detection of levels less than 1 mg/L.

Sample matrix effects in micellar electrokinetic capillary electrophoresis

Several factors related to sample matrix which can influence peak height in micellar electrokinetic capillary chromatography were studied. The ionic strength of the sample did not affect greatly the peak height. High concentration of surfactants or organic solvents in the sample decreased the peak height. On the other hand, using a surfactant in the sample different from the one in the electrophoresis buffer or the addition of polyethylene glycol to the sample enhanced slightly the peak height. A high surfactant concentration in the buffer increased the migration time as well as the plate number and the peak height. Matrix effects are more profound with large than with small sample injections. In general, the effect of sample matrix in MECC is much less than that observed in capillary zone electrophoresis. It is recommended to prepare the standards in the same matrix as that of the sample or to add the analytes directly to the sample to avoid any bias in the results.

Glycolate and 2-phosphoglycolate content of tissues measured by ion chromatography coupled to mass spectrometry.

Glycolate and 2-phosphoglycolate (PG) are 2-carbon monocarboxylic acids with ill-defined metabolic roles. Their concentrations have not yet been described in tissues apart from body fluids and erythrocytes. We describe the use of ion chromatography coupled with mass spectrometry (IC-MS) to quantify levels of glycolate and PG in tissue. Sample preparation and analysis can be performed within an hour. Low concentrations of glycolate (12-48 nmol/g) and PG (4-17 nmol/g) were detected in all tissues. The availability of this IC-MS assay will facilitate investigations of the origin, function, and metabolism of glycolate and PG in tissues.