Strategies for the monitoring of drugs in body fluids by micellar electrokinetic capillary chromatography

Electrokinetic capillary techniques can exploit numerous separation principles, making them flexible and easily applicable to a variety of separation problems. In recent publications, this emerging technology has been shown to be well suited for monitoring drugs and metabolites in body fluids, including serum, saliva and urine. Most attention has been focused on micellar electrokinetic capillary chromatography (MECC) because it permits the separation and determination of drugs with discrimination being largely based on differences in hydrophobicity. An overview of literature data on the MECC of drugs in body fluids and recent data obtained with antiepileptics in serum and saliva, with model mixtures of illicit drugs, and with extracts from urine specimens that tested positively for opiates and cocaine metabolites are presented. Emphasis is focused on buffer selection and simple sample preparation procedures, including direct injection of body fluids, ultrafiltration and solid-phase extraction.     

Analysis of recombinant cytokines in human body fluids by immunoaffinity capillary electrophoresis

An immunoaffinity capillary electrophoresis (ICE) system for rapidly quantifying recombinant cytokines in human body fluids has been developed. Cytokines within biological fluids were labeled with a red light emitting fluorochrome and injected into the capillary. Selected cytokines were captured by immobilized antibodies on the internal surface of the capillary, and held while unbound materials were purged. The cytokines were then eluted electrophoretically in acidic buffer. Individual cytokine peaks were detected by on-line laser-induced fluorescence detection coupled to a computerized fiber-optic spectrometer, and analyzed by integration of the eluted peaks. The comparison of the results of ICE to routine assays used for these cytokines demonstrates that ICE provides a fast and accurate procedure for defining these cytokines in complex biological samples. Immunoaffinity separations can be used for any material to which a specific antibody can be raised, making this procedure applicable to a wide range of molecules of biomedical interest.     

Distribution of free D-amino acids in tissues and body fluids of vertebrates

Reports on the distribution, metabolism and origins of free D-amino acids in vertebrate tissues and body fluids are reviewed. The transient emergence of D-aspartic acid during the development of brain and peripheral organs or early stages of life is reported. D-Serine in brain is postulated to be a potentiator for the N-methyl-D-aspartate (NMDA) receptor. Some D-amino acid concentrations in human serum, such as D-Ser and D-Ala, are suggested to correlate with damage to renal function.     

Determination of amino acids in biological fluids by capillary gas chromatography with nitrogen-phosphorus selective detection

A selective and sensitive method for the determination of protein and non-protein amino acids in biological fluids by capillary gas chromatography (GC) has been developed. The amino acids in the samples were directly converted into their N(O,S)-isobutoxycarbonyl methyl ester derivatives and measured by GC with nitrogen-phosphorus selective detection (NPD) using a DB-17ht capillary column. Using this method, the derivatives of the 21 protein amino acids and the 25 non-protein amino acids provided excellent NPD responses and were quantitatively and reproducibly resolved within 28 min. The lower detection limits of these amino acids, at a signal-to-noise ratio of 3, were ca. 6-150 pg injected. The calibration curves for each amino acid in the range of 0.02-2 micrograms were linear and sufficiently reproducible for quantitative analysis. This method was successfully applied to small urine and serum samples without prior clean-up; there was no evidence of interference from coexisting substances. Overall recoveries of amino acids added to urine and serum samples were 83-112%. The intra-assay and inter-assay R.S.D. of amino acids in these samples were 0.3-8.9% (n = 3) and 1.9-15.8% (n = 3), respectively.     

Nonaqueous capillary electrophoresis--its applicability in the analysis of food, pharmaceuticals and biological fluids

The use of nonaqueous electrophoresis media for the application of capillary electrophoresis in the analysis of food, pharmaceuticals and biological fluids is reviewed. Some of the applications are discussed in detail and the benefits of using nonaqueous media in these cases are outlined. Three new applications within pharmaceutical analyses are presented. In these methods either a simple sample pretreatment by dilution with methanol (determination of chlorhexidine in a cream) or selective on-line capillary electrophoresis mass spectrometry (methods for identification of seizure drugs or opium alkaloids) are used. The choice of organic solvents and electrolytes for nonaqueous capillary electrophoresis are discussed. Furthermore, validation data obtained using capillary electrophoresis based on the nonaqueous principle are listed and discussed.     

Use of the BacT/Alert blood culture system for culture of sterile body fluids other than blood

Studies have demonstrated that large-volume culture methods for sterile body fluids other than blood increase recovery compared to traditional plated-medium methods. BacT/Alert is a fully automated blood culture system for detecting bacteremia and fungemia. In this study, we compared culture in BacT/Alert standard aerobic and anaerobic bottles, BacT/Alert FAN aerobic and FAN anaerobic bottles, and culture on routine media for six specimen types, i.e., continuous ambulatory peritoneal dialysate (CAPD), peritoneal, amniotic, pericardial, synovial, and pleural fluids. Specimen volumes were divided equally among the three arms of the study. A total of 1,157 specimens were tested, with 227 significant isolates recovered from 193 specimens. Recovery by method was as follows: standard bottles, 186 of 227 (82%); FAN bottles, 217 of 227 (96%); and routine culture, 184 of 227 (81%). The FAN bottles recovered significantly more gram-positive cocci (P < 0.001), Staphylococcus aureus (P = 0.003), coagulase-negative staphylococci (P = 0.008), gram-negative bacilli (P < 0.001), Enterobacteriaceae (P = 0.005), and total organisms (P < 0.001) than the routine culture. There were no significant differences in recovery between the standard bottles and the routine culture. The FAN aerobic bottle recovered significantly more gram-positive cocci (P < 0.001), S. aureus isolates (P < 0.001), coagulase-negative staphyococci (P = 0.003), and total organisms (P < 0.001) than the standard aerobic bottle, while the FAN anaerobic bottle recovered significantly more gram-positive cocci (P < 0.001), S. aureus isolates (P < 0.001), Enterobacteriaceae (P = 0.03), and total organisms (P < 0.001) than the standard anaerobic bottle. For specific specimen types, significantly more isolates were recovered from the FAN bottles compared to the routine culture for synovial (P < 0.001) and CAPD (P = 0.004) fluids. Overall, the FAN bottles were superior in performance to both the standard bottles and the routine culture for detection of microorganisms from the types of sterile body fluids included in this study.     

Application of flow injection analysis in trace element determination of body fluids

Nucleophiles activated the catalytic actions of beta-galactosidases with neutral or positively charged substitutions for Glu-461. Aliphatic carboxylic acids increased the rate of hydrolysis of o-nitrophenyl beta-D-galactopyranoside if the pKa values of the carboxyl groups were > approximately 3.5. Amino compounds activated if their pKa values were < approximately 8.5. Imidazole, azide, and 2-mercaptoethanol also activated. Nucleophiles with high pKa values were able to activate the catalysis if the pH was high, and this showed that the lack of activation at pH 7.0 was because of protonation. Kinetic analysis showed that most of the nucleophiles that activated were bound to the active site, since the activation followed Michaelis-Menten type saturation kinetics. The binding seemed to be dependent upon the hydrophobicity; the longer the aliphatic chain, the stronger the binding. Gas-liquid chromatographic analysis showed that adducts of some type were formed during the reactions in the presence of many of the nucleophiles. Three of these adducts were purified and the nucleophiles were found beta-linked to D-galactose. This indicates that if an intermediate covalent bond is formed in the mechanism of beta-galactosidase action and if the nucleophile reacts to displace it, the intermediate covalent bond must have the alpha configuration and involve a group other than Glu-461.     

Use of reversed polarity and a pressure gradient in the analysis of disaccharide composition of heparin by capillary electrophoresis

A capillary electrophoresis method with reversed polarity, combining both the application of a voltage and a pressure gradient between the buffer vials, was developed for the analysis of eight heparin-derived delta-disaccharides obtained by enzymatic depolymerization. A 60 mM formic acid buffer at pH 3.40 was selected as running electrolyte, with an applied voltage of -15 kV and an over-imposed pressure gradient (3.45.10(-3) MPa) for 6 min from inlet to outlet starting at 20 min. Figures of merit such as run-to-run and day-to-day precision, and limits of detection were established. The electrophoretic method was applied to the analysis of depolymerization products of different kinds of heparins. The composition of the depolymerization buffer was selected in order to reduce baseline distortions in the electrophoretic separation, thus a buffer solution containing 20 mM Tris, 50 mM sodium chloride, and 3 mM calcium chloride at pH 7.10 was used. Percentages of molar disaccharide compositions for unfractionated heparins from porcine, bovine and ovine intestinal mucosa, and bovine lung were determined. In addition, low-molecular-mass heparins from bovine and porcine intestinal mucosa were analysed as well.     

Extracellular calmodulin-binding proteins in body fluids of animals

We aimed to audit nosological inaccuracies in death certification in Northern Ireland and to compare performance of hospital doctors and general practitioners. Nosology is the branch of medicine which treats of the classification of disease. 1138 deaths were registered in Northern Ireland in a 4-week period commencing 3/10/94. 195 of these were either registered by HM Coroners (HMC) or required further investigation by their staff; these cases were excluded from the study. The remaining 943 were analysed for wording and formulation inaccuracies according to the revised notes (1974), Northern Ireland Medical Certificate of Cause of Death. These are issued in book form by the Registrar of Births and Deaths. The commonest inaccuracies in death certification occur in the areas of poor terminology, sequence errors and unqualified mode. One or more inaccuracies were found in 317 (33.6%) of cases. In 13 of these (4%) cases, the inaccuracies were serious enough to warrant referral by the Registrar of Deaths to HM Coroner. The numbers of general practitioners and hospital doctors were recorded, with general practitioners being responsible for 122 (38%) and hospital doctors being responsible for 195 (62%) of inaccuracies.     

Amino acid analysis by gas-liquid chromatography of N-heptafluorobutyryl isobutyl esters. Complete resolution using a support-coated open-tubular capillary column

Amino acids have been separated by gas-liquid chromatography as their N-heptafluorobutyryl isobutyl esters. Complete resolution of derivatives of all the common amino acids has been achieved using a high-performance support-coated open-tubular capillary column. The analysis time was 30 min. Modifications to the derivatization procedure of MacKenzie and Tenaschuk have been introduced. Acylation by heating at 150 degrees was shown to be destructive; 110 degrees has been selected for routine preparation. To obtain a volatile histidine derivative it has been found necessary to add an antioxidant and to heat samples with ethoxyformic anhydride prior to injection. Amino acid analysis of beta-lactoglobulin after 6 N HCl digestion yielded results in good agreement with those obtained by the conventional ion-exchange method. The method has also been successfully applied to estimation of the different caseins in whole casein and in purified fractions by amino acid analysis of residues liberated by carboxy-peptidase digestion.     

Micromethod for analysis for chloride in physiological fluids

A micromethod for the analysis for chloride, based on the chemical precipitation of silver chloride by radial diffusion through agar gel containing silver nitrate, is described. The method is simple to run, requires little or no instrumentation, and requires only 10 mul of sample. Results by coulometric titration (Buchler Cotlove Chloridometer) correlated well for serum (r = 0.961), urine (r = 0.997), cerebrospinal fluid (r = 0.991), and sweat (r = 0.998). Other halide ions or protein do not interfere. Precision studies gave a within-day reproducibility (CV) of 1.3% and a day-to-day variability of 2.1% for a serum sample averaging 115 mmol/liter.     

Analysis of the components of Lycopus europaeus L. in body fluids during metabolism studies. Comparison of capillary electrophoresis and high-performance liquid chromatography

During pharmacokinetic studies with extracts obtained from medicinally used plants, analysis in body fluids is mainly performed by HPLC, an established separation method. In this paper high-performance capillary electrophoresis (HPCE) is investigated for its ability to separate such complex extracts. Crude extracts of Lycopus europaeus L. (Lamiaceae) are traditionally used against mild forms of hyperthyroidism. The metabolism of a 70% ethanolic extract with respect to some of its individual main components (rosmarinic and caffeic acid, luteolin-7-glucoside) and a mixture of the pure compounds were investigated using isolated perfused rat liver. After solid-phase extraction metabolites were determined using HPCE and HPLC separation techniques. A buffer solution composed of 0.05 mol l-1 Na2HPO4 at pH 7.0 with 30% acetonitrile was found to be the most suitable electrolyte for HPCE separation. The best mobile phase for isocratic HPLC was 0.03% TFA-acetonitrile (82:18, v/v). Data obtained with HPCE are in good accordance with those from HPLC; HPCE, however, is clearly more rapid and simple to perform.     

Use of principal component analysis for the evaluation of the retention behaviour of monoamine oxidase inhibitory drugs on beta-cyclodextrin column

The retention of 17 monoamine oxidase inhibitory drugs (proparlgylamine derivatives) were determined on a beta-cyclodextrin polymer (beta CDP)-coated silica column using ethanol-0.05 M K2HPO4 (6:4 v/v) as the eluent. The relative strength of interaction between the drugs and a water soluble beta-cyclodextrin polymer was determined by charge-transfer chromatography carried out on reversed-phase TLC layers. The relationship between capacity factors, physicochemical parameters and inclusion complex forming capacity of the monoamine oxidase inhibitory drugs were evaluated by stepwise regression analysis and by principal component analysis (PCA) followed by two-dimensional nonlinear mapping and varimax rotation. Calculations indicated that the retention of monoamine oxidase inhibitory drugs on beta CDP column is mainly governed by their steric and lipophylic parameters. Significant linear correlations were found between the corresponding coordinates of varimax rotation and two-dimensional nonlinear maps proving the suitability of both methods for the reduction of dimensionality of complicated data matrices.     

Use of capillary electrophoresis as a method development tool for classical gel electrophoresis

Capillary electrophoresis (CE) was used to optimize the buffer pH, ionic strength and sulfated cyclodextrin concentrations for enantiomeric separation of piperoxan. These enantioseparation conditions were then applied to a classical gel electrophoresis system. Binding constants of the sulfated beta-cyclodextrin-piperoxan couple were approximated using CE and the effects of organic solvents on the system were also investigated.     

Use of capillary electrophoresis in the study of ligand-DNA interactions

The presence and genetic content of integrons were investigated for 37 epidemiologically unrelated multiple-drug-resistant strains of Salmonella enterica serotype Typhimurium from humans. All isolates were resistant to ampicillin, chloramphenicol, kanamycin, streptomycin, sulfonamides, and trimethoprim, as well as to tetracycline and/or nalidixic acid; 20% of them were also resistant to gentamicin and amikacin. Three different class 1 integrons (In-t1, In-t2, and In-t3) were identified by Southern blot hybridization, PCR, and DNA sequencing, and these integrons were found to carry the aadB, catB3, oxa1, aadA1a, aacA4, and aacC1 gene cassettes. Integrons In-t1 (aadB and catB3) and In-t2 (oxa1 and aadA1a) were both located on a conjugative IncFI plasmid of 140 kb. In-t3 (aacA4, aacC1, and aadAIa) was located on an IncL/M plasmid of 100 kb which was present, in association with the IncFI plasmid, in gentamicin- and amikacin-resistant isolates. Despite the extensive similarity at the level of the antibiotic resistance phenotype, integrons were not found on the prototypic IncFI plasmids carried by epidemic Salmonella strains isolated during the late 1970s. The recent appearance and the coexistence of multiple integrons on two conjugative plasmids in the same Salmonella isolate are examples of how mobile gene cassettes may contribute to the acquisition and dissemination of antibiotic resistance.     

Analysis of compounds containing carboxyl groups in biological fluids by capillary electrophoresis

Capillary electrophoresis (CE) is one of the suitable separation techniques used to analyze drugs or metabolites in complicated sample matrices such as plasma, serum and urine. It sometimes requires only a simple process of sample pretreatment, deproteinization, dilution or extraction for biological fluids, otherwise no pretreatment is necessary. Various metabolic disorders concerning the compounds which possess carboxyl groups such as organic acids have been monitored by CE. Drug metabolism in the body can be monitored by the same technique. Recent publications suggest the feasibility of an automated system for diagnosis based on CE technique.