Capillary electrophoresis analysis of nitrite and nitrate in sub-microliter quantities of airway surface liquid.

We developed a simple capillary electrophoresis (CE) method to measure nitrite and nitrate concentrations in submicroliter samples of rat airway surface liquid (ASL), a thin (10-30 microm) layer of liquid covering the epithelial cells lining the airways of the lung. The composition of ASL has been poorly defined, in large part because of the small sample volume (approximately 1-3 microl per cm2 of epithelium) and difficulty of harvesting ASL. We have used capillary tubes for ASL sample collection, with microanalysis by CE using a 50 mM phosphate buffer (pH 3), with 0.5 mM spermine as a dynamic flow modifier, and direct UV detection at 214 nm. The limit of detections (LODs), under conditions used, for ASL analysis were 10 microM for nitrate and 30 microM for nitrite (SIN= 3). Nitrate and nitrite were also measured in rat plasma. The concentration of nitrate was 102+/-12 microM in rat ASL and 70+/-1.0 microM in rat plasma, whereas nitrite was 83+/-28 microM in rat ASL and below the LOD in rat plasma. After instilling lipopolysaccharide intratracheally to induce increased NO production, the nitrate concentration in ASL increased to 387+/-16 microM, and to 377+/-88 microM in plasma. The concentration of nitrite increased to 103+/-7.0 microM for ASL and 138+/-17 microM for plasma.

Approaches to quantitative structure-enantioselectivity relationship modeling of chiral separations using capillary electrophoresis.

Quantitative structure-enantioselectivity relationships (QSERs) have been developed to describe the resolution of a series of chiral arylpropionic acids using capillary electrophoresis. Native beta-cyclodextrin and two derivatized forms are used as the chiral resolving agents. The QSER models are developed using the results of molecular mechanics calculations as input to multivariate linear regression and also to neural networks. Single models are developed to predict the optimum cyclodextrin to resolve a given analyte, the migration order, and the magnitude of the separation. Models are also developed to predict only the optimum cyclodextrin.

Analysis of small-scale biological compartments by capillary electrophoresis.

Two characteristics of capillary electrophoresis make the technique attractive for the separation of the components of microscale compartments within living organisms: small sample volume requirements and direct compatibility with biofluids. Indeed, capillary electrophoresis has been used for analysis down to a sub-cellular level. There are also potentially many applications of capillary electrophoresis to biological compartments on a super-cellular scale, which are nevertheless so small that they make analysis by conventional separations techniques difficult or impractical. The analytical challenges in small-scale bioanalysis are first to develop a suitable method for collection of sample and its introduction into the separation capillary, and secondly, to achieve the required separation. Examples reviewed here will primarily focus on the analysis of tear fluid or airway surface liquid, cases in which the amount of sample that can be collected range from around 10 microl to around 100 nl.

Automated sample preparation of Roxifiban tablets: transfer of a manual method to an automated workstation.

Automation offers obvious advantages for the preparation of tablets prior to analysis by HPLC including unattended operation, minimization of human intervention and an electronic audit trail. However, significant effort has to be put in up front to develop and validate an automated method, particularly if it is required to closely follow an existing manual method. Here, method transfer for Roxifiban, a fibrinogen receptor antagonist, will be discussed. A Zymark tablet processing workstation II (TPWII) was used for all automated sample preparations. Manual methods for composite assay, content uniformity, weight variation and degradation products testing of a tablet formulation were transferred to the TPWII. The method involved weighing of the sample, disintegration of the dosage form by homogenization, extraction of the analyte in the homogenate solution, filtration of the homogenate, dilution of the filtrate and transfer to autosampler vials. Obstacles to a quick transfer included limitations in the volume capabilities of the TPWII, poor analyte solubility and achieving proper conditioning of the transfer lines and filter. After resolving these issues, a validated method was achieved. Spiked recoveries were from 99.4 to 101.1% (RSD's <0.5%). A cross-validation between automated and manual assay methods was compared by Westlake analysis giving a 0.7% calculated interval at the 95% confidence level. Carryover was 0.07% after 20 sample preparations at the highest tablet strength.

The effect of stationary-phase pore size on retention behavior in micellar liquid chromatography.

One of the limitations that has restricted the applicability of micellar liquid chromatography (MLC) is the weak eluting power of micellar mobile phases compared to conventional hydro-organic mobile phases used in reversed-phase liquid chromatography. This may be the result of Donnan or steric exclusion of the micelles from the pores of the stationary phase, within which nearly all (> or = 99%) of the stationary phase resides and the analytes spend most of their time. To determine whether wide-pore stationary phases would overcome this limitation in MLC, several C8 and C18 stationary phases ranging from 100 to 4000 A were investigated using a diverse set of test solutes and micellar solutions of anionic, neutral, and cationic surfactants as mobile phases. With the larger pore size stationary phases, the eluting power of the MLC mobile phases was enhanced with all surfactant types, the greatest effect being with the neutral surfactant. Differences in retention behavior were observed between various solute types and between the C8 and C18 stationary phases. These differences appear to be related to the relative hydrophobicity of the solutes and to differences in the surfactant-modified stationary phases. Partitioning behavior of representative solutes on the large-pore C8 and C18 columns was shown to follow the three-phase partitioning model for MLC. Methylene group selectivity data showed only minor differences in the stationary-phase characteristics between the small- and large-pore size C18 columns. The true eluting power of micellar mobile phases was revealed with wide-pore stationary phases and was demonstrated by the separation and elution of an extended series of alkylphenones on C18 columns.

Radiosensitization of a mouse tumor model by sustained intra-tumoral release of etanidazole and tirapazamine using a biodegradable polymer implant device.

BACKGROUND AND PURPOSE: Drug toxicities are often a limiting factor in long term treatment regimes used in conjunction with radiotherapy. If the drug could be localized to the tumor site and released slowly, then optimal, intra-tumoral drug concentrations could be achieved without the cumulative toxicity associated with repeated systemic drug dosage. In this paper we describe the use of a biodegradable polymer implant for sustained intra-tumoral release of high concentrations of drugs targeting hypoxic cells. MATERIALS AND METHODS: The RIF-1 tumor was implanted subcutaneously or intramuscularly in C3H mice and irradiated with 60Co gamma rays. The drug delivery device was the co-polymer CPP-SA;20:80 into which the drug was homogeneously incorporated. The hypoxic radiosensitizer Etanidazole or the bioreductive drug Tirapazamine were delivered intra-tumorally by means of implanted polymer rods containing the drugs. Tumor growth delay (TGD) was used as the end point in these experiments. RESULTS: Both Etanidazole and Tirapazamine potentiated the effects of acute and fractionated radiation in the intra-muscular tumors but neither drug was effective in sub-cutaneous tumors. Since both drugs target hypoxic cells we hypothesized that the lack of effect in the subcutaneous tumor was attributable to the smaller size of the hypoxic fraction in this tumor model. This was confirmed using the hypoxia marker EF5. CONCLUSIONS: These results indicate that the biodegradable polymer implant is an effective vehicle for the intra-tumoral delivery of Etanidazole and Tirapazamine and that, in conjunction with radiation, this approach could improve treatment outcome in tumors which contain a sub-population of hypoxic, radioresistant cells.

Cisplatin delivery by biodegradable polymer implant is superior to systemic delivery by osmotic pump or i.p. injection in tumor-bearing mice.

The use of biodegradable polymer implants to deliver cisplatin was compared with delivery by systemic injection and by osmotic pump. Drug levels in the tumor were found to be higher than those in the blood and kidney when the drug was delivered using the polymer implant. In contrast, for the other two delivery methods blood and kidney cisplatin levels were greater than those in the tumor. It has been previously shown that tumor response, in terms of growth delay, was greatest when drug was delivered by polymer implant and least when treatment was by osmotic pump.

The potentiation of the effect of radiation treatment by intratumoral delivery of cisplatin.

PURPOSE: To compare potentiation of the effects of acute or fractionated radiation by cisplatin when the drug was delivered intratumorally by implanted biodegradable polymer, by intraperitoneal injection, or by intraperitoneal osmotic pump. METHODS AND MATERIALS: Radiation was delivered to a mouse tumor (RIF-1) either in a single dose or in a fractionated regime in conjunction with cisplatin delivered either as a bolus injection, with an osmotic pump, or with a biodegradable polymer rod containing cisplatin. The osmotic pump was implanted in the intraperitoneal cavity of the mouse while the polymer implants were placed directly in the tumor. As the polymer degrades, the drug is released at the treatment site leading to high local concentrations. The osmotic pump, in contrast, leads to prolonged systemic exposure to the drug at low concentrations. Tumor growth delay (TGD) was used as an endpoint in these experiments. RESULTS: The most effective treatment protocol, in terms of potentiating the effects of radiation was cisplatin delivered by polymer implanted 2 days before an acute dose of radiation (growth modification factor [DMF] = 2.2). Comparison of single and multifraction regimes where polymer implant was on the same day as the commencement of treatment showed greater potentiation of the effect of fractionated than of acute radiation treatment with the DMF for fractionated treatment remaining relatively constant (1.5-1.9) for 5, 8, and 12 fraction treatments. Cisplatin delivered via the osmotic pump did not deliver a high enough dose of cisplatin to produce therapeutic effect in this mouse tumor model and had little impact on response to treatment. CONCLUSIONS: Our results indicated that cisplatin delivered intratumorally by biodegradable polymer implant was effective in potentiating the effect of both acute and fractionated radiation. For the fractionated treatments the effect was maintained with increasing fraction numbers and treatment time.

Evaluation of beta-lactoglobulin as a stationary phase in high-performance liquid chromatography and as a buffer additive in capillary electrophoresis: observation of a surprising lack of stereoselectivity.

Previous studies have reported that alpha1-acid glycoprotein is quite similar in amino acid sequence and disulfide bond arrangements to members of a group of proteins which include beta-lactoglobulin (BLG). Since generally homologous proteins retain some similarity in function at the molecular level, we decided to evaluate the enantioselective properties of BLG as an high-performance liquid chromatographic chiral stationary phase (HPLC-CSP), and as an additive in capillary electrophoresis (CE). Two columns with differences in internal diameter and method of immobilisation on epoxide silica were prepared. Chiral acidic, basic and uncharged drugs were chromatographed and mobile phase parameters, namely pH and type of organic modifier, were varied in order to test the column performance. The CE approach has some advantages in that there is no need for immobilisation and only a small amount of protein is required. BLG was therefore tested as a CE buffer additive, using the same analytes as in the HPLC study. Although one would expect that a protein would display some enantioselectivity, BLG did not show any enantioselectivity whatsoever in either system; the protein has fairly weak interaction with the majority of the test solutes, as indicated by both techniques.

Analysis of proteins in microsamples of rat airway surface fluid by capillary electrophoresis.

A thin layer of airway surface fluid (ASF) lining the pulmonary airways plays an important role in the primary defense mechanisms of the lung against bacterial infection. However, little is known about the composition of ASF due to the thinness (typically 5-30 microm in healthy animals) of the fluid layer and its relative inaccessibility, which causes considerable difficulties in sample collection and subsequent analysis. We have used a novel technique of capillary sampling coupled with capillary electrophoresis (CE) to analyze the protein composition of rat ASF. CE analyses were performed under two different conditions: a borate buffer, pH 9.1, or a phosphate buffer, pH 2.5, with 0.5 mM spermine. The different selectivities afforded by the two methods aid in peak identification, and quantitation of most of the major species was possible using both separation conditions. Albumin, transferrin and globulins are observed to be the major protein components in rat ASF, at concentrations of 28 mg ml(-1), 4.0 mg ml(-1) and 34 mg ml(-1) respectively, in comparison to 31 mg ml(-1), 3.1 mg ml(-1) and 40 mg ml(-1), respectively, in rat plasma.

Airway surface fluid composition in the rat determined by capillary electrophoresis.

The apical surface of respiratory epithelial cells is covered by a thin layer of low-viscosity fluid termed airway surface fluid (ASF), about which relatively little is known. We collected samples of ASF from anesthetized rats, which were then analyzed using capillary electrophoresis, a method that enables extremely small quantities of fluid to be analyzed. We found values for Na+ (40.57 +/- 3.08 mM), K+ (1.74 +/- 0.36 mM), and Cl- (45.16 +/- 1.81 mM), indicating that this fluid is hypotonic compared with rat plasma. In contrast, the concentrations of nitrite and nitrate within ASF were higher than reported plasma values. Additionally, intravenous administration of the cholinergic agonist methacholine (MCh) resulted in a dose-dependent increase in the concentration of Na+ and Cl- within the ASF. This increase is approximately 50% in these ions after a dose of 100 ng MCh/g body wt. This animal model, together with this microanalytical technique, may be useful for investigating the in vivo regulation of ASF composition.

Determination of p-aminosalicylic acid and its N-acetylated metabolite in human urine by capillary zone electrophoresis as a measure of in vivo N-acetyltransferase 1 activity.

A capillary zone electrophoresis method has been developed for the determination of p-aminosalicylic acid (PAS) and its metabolite, N-acetyl-p-aminosalicylic acid (N-acetyl-PAS), in urine. A linear relationship was observed between time-normalized peak area and the concentration of the parent and metabolite with correlation coefficients greater than 0.9990. The method could be applied to the determination of PAS and N-acetyl-PAS in human urine without any sample pretreatment. A good separation of the analytes is achieved in a run time of 12 min (15 min total, including capillary wash). Using PAS as a probe for N-acetyltransferase 1 activity, 20 healthy volunteers were phenotyped after oral administration of a 1 g dose. The preliminary results seem to indicate a bimodal distribution of N-acetyl-PAS/PAS molar ratios.

Tumor treatment by sustained intratumoral release of cisplatin: effects of drug alone and combined with radiation.

PURPOSE: The effect of intratumoral delivery of cisplatin to a mouse tumor model (RIF-1) by means of a biodegradable polymer implant with and without radiation was studied. METHODS AND MATERIALS: The polymer bis (p-carboxyphenoxy) propane-sebacic acid (CPP:SA; 80:20) and its degradation products have been characterized. Polymer rods (8 x 0.5 mm) containing 17% cisplatin by weight were prepared by extrusion, and the in vitro degradation rate measured. The implants were placed into mouse tumors and their effect (with and without radiation) on tumor growth delay studied. The levels of Pt in the mouse kidney, tumor, and blood plasma at selected intervals after implant were also determined. These results were compared with those obtained when cisplatin was delivered systemically. RESULTS: When cisplatin was delivered by the polymer implants, higher levels were present in the tumor for longer time periods (cf. systemic delivery of the drug). For both nonirradiated and irradiated tumors, those treated with the polymer implants had significantly longer tumor growth delays compared to nonimplanted controls and to systematically treated tumors. CONCLUSIONS: The results show that intratumoral delivery of cisplatin is more efficient than systemic delivery. Using the biodegradable polymer implant, higher doses of cisplatin can be tolerated by the animal as the drug is localized within the tumor, and the high levels of the drug in the tumor can be maintained for an extended period of time. When radiation is given in conjunction with cisplatin, the tumor response is supraadditive for all modes of cisplatin administration but is potentiated to a greater extent when cisplatin is delivered through the polymer implant. The greatest effect is seen for treatment with cisplatin delivered by polymer implant combined with fractionated radiation.

Microanalysis of lung airway surface fluid by capillary electrophoresis with conductivity detection.

The thin layer of fluid that covers the surface of the epithelia lining the conducting airways plays an important role in primary pulmonary defense, and its composition may be a critical factor in the pathogenesis of several lung diseases including cystic fibrosis. Despite its physiological importance, the composition of airway surface fluid (ASF) is poorly understood due to considerable difficulties in sample collection from the 5-30 microns thick layer and subsequent analysis. We have used a novel technique for sample collection and microanalysis of ASF (nanoliter sample required) by capillary electrophoresis with conductivity detection. Limitations on the diameters of capillary required for the sample injection process and for the conductivity detector require the use of coupled separation capillaries with different external diameters. Two different methods were used to construct a butt-joint coupling for capillaries of different outer diameters. Reasonable efficiency is observed with the coupled capillaries (N = 100000 plates m-1) compared to an unbroken single capillary (N = 180000 plates m-1). The use of conductivity detection allows greater flexibility in method development and the possibility of determining a greater variety of ions than with a previous indirect-UV method. In the present study, we describe the analysis of cations (Na+, K+, Ca2+, Mg2+) and anions (Cl-, NO2-, NO3-, SO4(2-), PO4(2-), HCO3-) in rat ASF. Particular attention was paid to developing washing procedures which limited fouling of the conductivity sensor. In healthy rats, ASF was found to be hypotonic compared to plasma levels, consistent with some observations made in human airways.

A quantitative relationship between capacity factor and selector concentration in capillary electrophoresis and high performance liquid chromatography: evidence from the enantioselective resolution of benzoin using human serum albumin as a chiral selector.

Many selectors are used both in pressure-driven liquid chromatography (LC) and in electrokinetic chromatography (EKC), particularly chiral species such as cyclodextrins and proteins. It should be possible to readily apply information gleaned using one technique to the other, since in both techniques the underlying molecular interactions which lead to separations are expected to be the same. Superficially this may be the case, but an exact transfer of operating conditions, i.e., background electrolyte (BGE) composition/mobile phase composition, assuming that these meet certain minimum requirements for each technique, is not often possible. To investigate the reason for this we have measured retention (k') of a neutral solute (racemic benzoin) in HPLC and EKC using an identical range of BGE/mobile phase conditions in both techniques. The selector used was human serum albumin. The k' measurements obtained for each benzoin enantiomer were consistently higher in HPLC than in EKC. This can be explained very simply if one considers that retention in both systems is related to the selector concentration [S], by the expression k'=K[S], where K is the affinity constant. In EKC, [S] is simply the concentration of free selector in the BGE, while in LC, [S] = m(p)/Vo, where m(p) is the number of moles of accessible selector, and Vo is the column void volume. In LC, [S] is generally considerably higher than in EKC, leading to larger values of k'.

Effect of organic modifiers on retention and enantiomeric separations by capillary electrophoresis with human serum albumin as a chiral selector in solution.

We have investigated the effect of methanol, ethanol, 1-propanol, 2-propanol and acetonitrile on the retention and enantiomeric separations of benzoin and propiomazine by capillary electrophoresis, using human serum albumin as a chiral selector. The effects of these modifiers on mobilities of analytes are rather difficult to interpret. Calculation of capacity factors reveals the underlying analyte-protein interactions; pitfalls in making such calculations are pointed out. In the case of benzoin and propiomazine binding to human serum albumin, capacity factors were observed to always decrease upon addition of organic modifiers, although the effects of 1- and 2-propanol suggest a possible specific interaction or modification of the protein conformation.

Selectivity in capillary electrophoresis: the use of proteins.

Proteins, by their very diverse nature, provide a wide variety of options for generating selectivity in capillary electrophoresis (CE). Their use in different modes of CE will be considered in this review. Proteins added in solution to the background electrolyte allow separations to be made in a similar fashion to other electrokinetic chromatography methods, e.g., micellar separations. Alternatively, different immobilization schemes can be used to secure proteins within the capillary; these have included capillary electrochromatography with the protein grafted onto a silica support, or immobilization of the protein within a gel structure. Compounds varying in size from small inorganic ions to biopolymers may be bound by proteins. There is the potential for any sort of intermolecular interaction to play a role in the binding process (e.g., hydrophobic interactions, electrostatic interactions, etc.). Very specific high-affinity binding often occurs, but also there is often weaker, non-selective binding. Frequently the interactions of chiral compounds with proteins are stereoselective. Obtaining chiral selectivity has been one of the main applications of protein selectors in CE, and this use will be emphasized here in a discussion structured by type of protein. As well as utilizing the selectivity of proteins to develop separations, the role of CE in investigating ligand-protein interactions will be emphasized.

Capillary electrophoretic analyses of drugs in body fluids: sample pretreatment and methods for direct injection of biofluids.

A variety of strategies for the analysis of biological samples by capillary electrophoresis (CE) are described, with particular emphasis on the determination of drugs and metabolites. Analytical methods involving extensive sample pretreatment before CE analysis are considered, as well as strategies for directly injecting untreated biofluids. The application in CE of techniques common in liquid chromatography is first described, e.g. protein precipitation, liquid-liquid extraction and solid-phase extraction. On-capillary methods of sample concentration are considered. Approaches to performing CE assays of urine and plasma, without prior sample treatment, are described. The use of both capillary zone electrophoresis and micellar electrokinetic chromatography for direct-injection assays is compared for both urine and plasma analyses, and capillary washing strategies are discussed. Finally, direct-injection microanalyses are mentioned.

Separation of testosterone metabolites in microsomal incubates using a new capillary electrophoresis assay.

A capillary electrophoresis method has been developed to separate the products of liver microsomal testosterone metabolism. The microsomal mixture undergoes liquid-liquid extraction and pre-concentration, and then electrophoretic analysis takes less than 25 min including capillary conditioning steps. The development of the complex background electrolyte (Tris-HCl and borate buffers, sodium dodecyl sulfate, beta-cyclodextrin and ethanol) necessary for this separation is described. A z-type capillary flow cell is used to obtain adequate detection sensitivity. The proportion in which the metabolites are produced as determined by this method allows assignment of the relative activity of cytochrome P-450 enzymes in the microsomes. The technique is useful for comparison of activity in normal and abnormal hepatic microsomes.