Z. K. Shihabi

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.

Review of Drug Analysis with Direct Serum Injection on the HPLC Column

Abstract Contrary to the expectations of the past decade, routine therapeutic drug monitoring by HPLC failed to expand. Although more extensive to perform, immunoassays have dominated the market for drug analysis in the last few years. This is attributed to the lengthy sample preparation needed for HPLC. However, HPLC remains the main method for therapeutic drug monitoring of the uncommon drugs and their metabolites. The method of direct serum injection on the column simplifies HPLC as an analytical technique and enables full automation. Advantages and disadvantages of this method of analysis are discussed. This review tries to summarize the different approaches for direct serum injection on the HPLC column: 1) Guard columns, 2) Micellar chromatography, 3) Internal surface reversed-phase chromatography, 4) Column switching, and 5) Wide-pore (protein) columns. With direct serum injection, the pH of the mobile phase is very critical for separating the drug of choice from the endogenous substances found in s...

Stacking of weakly cationic compounds by acetonitrile for capillary electrophoresis

Abstract The inclusion of acetonitrile in the sample causes stacking especially in presence of sodium chloride. It is shown here that the type of the separation buffer and the conditions for the stacking of cationic versus anionic compounds are quite different. Anionic compounds stacked better in high ionic strength inorganic buffers such as borate and phosphate, while the cationic drugs and endogenous substances stacked better in amine and zwitterionic buffers. About 10–20% of the capillary volume can be loaded with sample. Shielding against the negative charges of the silica is a critical factor in stacking of the cationic compounds by the acetonitrile in the sample. The ionic strength of the separation buffer and the addition of organic modifiers affected the stacking to a limited extent.

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.

Serum lamotrigine analysis by capillary electrophoresis

Lamotrigine, a new antiepileptic drug, is analyzed by capillary zone electrophoresis. Samples were deproteinized with acetonitrile containing an internal standard, acidified with dilute acetic acid and injected into the capillary. The drug migrated rapidly with the cationic compounds in about 3.5 min far from any interfering substances. The test was linear between 0.5-10 mg/l. The analysis time was about 5 min. The CE values correlated well with an HPLC method (r = 0.97; n = 35). The mean serum concentration of 121 patients on this drug was 3.7 mg/l. Incubating the serum with beta-glucuronidase for 1 h increased the peak height of lamotrigine by about 24%.

Insulin stacking for capillary electrophoresis

Stacking methods are very important in overcoming the poor detection limits in capillary electrophoresis. Human insulin, a polypeptide, was concentrated on the capillary (stacked) based on three different and simple treatment methods to the sample: dilute buffers, high salt content, and acetonitrile (66%) were added to the sample to induce stacking. A dilute buffer in the sample caused a limited stacking, while acetonitrile treatment and high salt content in the sample caused much greater (approximately 20-fold) stacking. High salt concentration in the sample caused stacking presumably by a transient isotachophoretic method. In addition to stacking, the acetonitrile treatment removed the excess proteins in the sample. Insulin did not denature or precipitate in 66% acetonitrile as confirmed by high-performance liquid chromatography (HPLC) and immunoassays. Acetonitrile treatment enabled one-third of the capillary to be loaded with sample thus increasing the detection signal greatly. The insulin peak after acetonitrile treatment and separation by capillary electrophoresis (CE) was confirmed by HPLC and by CE fraction collection followed by immunoassay. Based on acetonitrile treatment, insulin detection in pancreatic tissue homogenates is shown to be feasible.

Therapeutic drug monitoring by capillary electrophoresis

Because of the ease of analysis and the high resolution, drug analysis is becoming the best example for the application of capillary electrophoresis. Therapeutic drug monitoring is a specialized area of drug analysis performed in clinical laboratories for patient care. CE offers high resolution and speed with the low operating costs needed in patient care. However, CE has a few limitations, mainly poor detection limits and precision. Simple methods of stacking, which enhance drug detection to overcome the poor sensitivity of CE are stressed. Serum has a unique matrix with a high content of proteins and salts which can have adverse effects on separation by CE. For successful analysis, special maneuvers are employed to decrease these matrix effects. Studies that have addressed the improvement of the precision of CE are summarized. CE offers the possibility of bringing chiral separations into the routine arena.

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.

Iohexol Determination by Direct Injection of Serum on the HPLC Column

Abstract Iohexol, a non-ionic compound used as a contrast media for angiography and as a mesure to determine glomerular filtration rate, was measured in serum by injecting serum directly on the column after a 200 fold dilution. The analysis was accomplished in less than 5 min.