Octyl Silica Column Research Articles

Development and validation of a liquid chromatography/electrospray ionization tandem mass spectrometry method for the quantification of latanoprost free acid in rabbit aqueous humor and ciliary body

A rapid, selective and sensitive method for quantification of latanoprost free acid in rabbit aqueous humor (AH) and ciliary body (CB) using reverse phase-high performance liquid chromatography coupled with electrospray ionization (ESI)-mass spectrometry/mass spectrometry has been developed and validated. Quantification in AH and CB was achieved by stable isotope dilution employing tetra-deuterated analog of latanoprost free acid, used as internal standard. Sample preparation was based on protein precipitation with methanol in AH, and on liquid extraction with a mixture of ethyl acetate and isopropanol 60:40 (v/v) in CB. Elution was achieved on an octylsilica (C8) column, using an isocratic elution method. Detection was performed on a triple quadrupole mass spectrometer, using ESI in positive ion selected reaction monitoring mode. Calibration curves were linear in the validated concentration ranges of 10-160 ng/mL in AH and 80-1280 ng/g in CB. The accuracy and precision values, obtained from three different sets of quality control samples, each analyzed in triplicate on three different days, were within the generally accepted criteria for analytical methods (< 15%). The limit of detection was 30.66 pg/mL in AH and 237.75 pg/g in CB. The assay proved to be accurate and precise when applied to the in vivo study of latanoprost free acid in rabbit AH and CB after single administration of an eye drops containing latanoprost.

Determination of methotrexate and indomethacin in urine using SPE-LC-DAD after derivatization

A validated HPLC-DAD assay is presented for the simultaneous quantification of methotrexate and indomethacin in a drug combination which is used synergistically to intervene with tumoral or inflammatory tissue microenvironments. The analytes were isolated from urine via solid phase extraction. The method is based on derivatizing both analytes with a soluble carbodiimide coupler and 2-nitrophenylhydrazine directed to their commonly occurring carboxylate functions. The chromatographic separation was accomplished on an octylsilica column in less than 15 min using acetate buffer (pH 4; 10 mM)–methanol (60:40, v/v) as eluent at 1.5 ml/min and monitored at 400 nm. The selectivity of the method was demonstrated in a pre-dosed rheumatoid arthritis patient. Quality control samples were prepared and analyzed to reveal the validity of the method. Life samples collected from a healthy volunteer were monitored for both drugs and their urinary levels were determined.

Presence of immunoreactive salusin-β in human plasma and urine

Salusin-α and salusin-β are multifunctional bioactive peptides originally identified using bioinformatics analyses. Salusin-β has been shown to exert potent hypotensive, bradycardic, and pro-atherosclerotic effects. The form in which it exists in biological fluids remains undetermined due to technical difficulties originating from its unexpected physicochemical properties. Here we show that salusin-β peptide adheres to polypropylene and polystyrene, so that the aliquoted peptide dissolved in distilled water may rapidly disappear from the solution. By circumventing these features and using an antibody against C-terminal portion of salusin-β, we have successfully established a specific radioimmunoassay suitable for detection of immunoreactive human salusin-β. We have characterized the molecular form of salusin-β in human plasma and urine. The assay detected immunoreactive salusin-β concentrations as low as 5 fmol/tube and the concentration required for 50% inhibition of binding was 122 fmol/tube. Cross-reactivities with salusin-α and other bioactive peptides were negligible. Reverse-phase high performance liquid chromatography coupled with the radioimmunoassay detection after extraction from plasma and urine and using an octyl-silica column, revealed a major immunoreactive component that co-eluted with authentic salusin-β. Salusin-β-like immunoreactivity in normal human urine ranged from 0.23 to 2.22 nmol/l (mean ± SD, 1.16 ± 0.84 nmol/l, n = 10). These data present the first evidence that salusin-β circulates and is excreted in its authentic form, thereby verifying the initially predicted processing sites for salusin-β in humans.

Expression of prosalusin in human neuroblastoma cells

Salusins, which are derived from the prosalusin precursor molecule, regulate hemodynamics, mitogenesis and atherogenesis. The preprosalusin gene is ubiquitously expressed, while the salusin-β peptide is present in systemic endocrine cells and the neuroendocrine system. However, the regulatory mechanisms for the preprosalusin gene and prosalusin expression remain to be investigated. Real-time quantitative RT-PCR and salusin-α radioimmunoassay revealed that the neuroblastoma cell line, SK-N-SH, exhibited marked upregulation of preprosalusin mRNA and salusin-α-like immunoreactivity (LI) when incubated under 2% serum condition. However, SK-N-SH cells released a limited amount of salusin-α-LI into the culture supernatant. Reverse-phase high performance liquid chromatography coupled with radioimmunoassay after extraction of proteins from the conditioned media using an octyl-silica column did not reveal a component that co-eluted with authentic salusin-α. Western blotting of the nuclear extracts from SK-N-SH showed the expression of prosalusin and its cleaved fragments, but not authentic salusin-α. Addition of Jak-2 inhibitors to growing SK-N-SH cells cultured under 10% serum condition resulted in increased salusin-α-LI expression. Suppression of Jak-2 mRNA using siRNAs upregulated intracellular salusin-α-LI, as detected by immunofluorescence. In summary, the preprosalusin gene and prosalusin protein are expressed in a neuroblastoma cell line and upregulated by reduced serum. The Jak-2 pathway may be involved in the regulation of salusin expression.

Determination of rifalazil in dog plasma by liquid–liquid extraction and LC–MS/MS: Quality assessment by incurred sample analysis

Incurred dog plasma samples were utilized for method quality assessment in this study, where a sensitive LC–MS/MS method was used for determination of the antibacterial agent rifalazil (KRM-1648 or ABI-1648). Reproducibility was estimated by repeated analysis of samples from a pharmacokinetic study, where 23 out of 864 study samples were reassayed during the course of the study. Precision for same-day duplicates was %R.S.D. 3.1 (concentration range 0.7–149 ng/ml), and over the whole study %R.S.D. 11.0 (concentration range 0.5–52 ng/ml). Moreover, standard addition experiments with incurred samples (concentration range 0.30–45 ng/ml) are described, where the recovery of spiked rifalazil amount was measured as a surrogate parameter for accuracy. The mean recovery of the added rifalazil amount was 103% (%R.S.D. 26.2). It was concluded that the described method is robust and reproducible for incurred samples. Liquid–liquid extraction was used for isolation of rifalazil and an isotope labeled internal standard from plasma. A manual procedure, based on an 8 × 12 array format, was used for sample extraction. Extracts were analyzed by reversed-phase liquid chromatography using octylsilica column with gradient elution (1 mM ammonium acetate + 0.01% (v/v) acetic aid in water and methanol). Mass spectrometric detection was made with positive ion electrospray ionization and LC–MS/MS analysis in a triple-quadrupole mass spectrometer. The lower limit of quantification was 50 pg/ml. MS characteristics of rifalazil are presented. In particular, two different sets of ionization and selected reaction monitoring (SRM) conditions, where in-source fragmentation was used for precursor ion formation in one of the sets, were compared. The good correlation found between the two sets of results for authentic sample extracts indicated that either condition could be used for quantification of rifalazil in dog plasma.

Presence of immunoreactive salusin-α in human serum and urine

Salusins, identified from a full-length enriched human cDNA library by bioinformatics analyses, show mitogenic, neuromodulatory and hemodynamic activities in rats. They are expressed in a wide variety of human tissues, but their precise structures and levels in human body fluids remain unknown. We developed a radioimmunoassay suitable for the detection of immunoreactive human salusin-α and characterized the molecular forms and concentrations of salusin-α in human serum and urine. The assay allowed for measurement of immunoreactive salusin-α concentrations as low as 1 fmol/tube after extraction of serum with an octyl-silica column, and the concentration required for 50% inhibition of binding was 40 fmol/tube. Cross-reactivities with salusin-β and other bioactive peptides were negligible. Salusin-α-like immunoreactivity in normal human serum and urine ranged from 11.0 to 40.4 pmol/l (mean ± S.D., 23.3 ± 8.1 pmol/l, n = 31) and from 18.6 to 367.3 pmol/l (mean ± S.D., 156.8 ± 95.8 pmol/l), respectively. Reverse-phase high performance liquid chromatography coupled with radioimmunoassay detection revealed a major immunoreactive component that coeluted with authentic salusin-α. These data indicate the presence of salusin-α in human serum and urine, thereby verifying the initially predicted processing sites for salusin-α in humans.

Benzotriazoles, Alkylphenols and Bisphenol A in Municipal Wastewaters and in the Glatt River, Switzerland

Many organic micropollutants occur at trace concentrations in municipal wastewater effluents and in the aquatic environment. Some of these xenobiotic chemicals can be considered as 'emerging' contaminants and some are suspect to have endocrine disrupting effects. Among the latter are nonylphenol (NP), octylphenol (OP) and bisphenol A (BPA), which deserve special attention due to their ubiquitous occurrence in the aquatic environment. The complexing agents benzotriazole (BT) and tolyltriazole (TT) are applied as anticorrosive agents (e.g. in cooling and hydraulic fluids, in antifreezing fluids, in aircraft deicing fluids, in dish washing liquids for silver protection), as antifogging agents and as intermediates for the synthesis of various chemicals. The environmental occurrence of NP and OP is caused by the fact that they are intermediate products (metabolites) in the biodegradation of alkylphenol polyethoxylate surfactants. BPA is globally used for the production of polycarbonate and epoxy resins. BT, TT, NP, OP and BPA were quantitatively determined in municipal wastewater effluents in Switzerland and in the Glatt River. The analytes were enriched by solid-phase enrichment. BT and TT were determined underivatized by electrospray LC/tandem MS. Reversed-phase LC was performed on octylsilica columns with isocratic water/methanol elution. Multiple reaction monitoring of the positive ions provided selective and sensitive detection for reliable quantifications. NP, OP and BPA were determined by GC/MS after derivatization with N-methyl-N-(trimethylsilyl)-trifluoroacetamide. BT and TT concentrations in primary and secondary effluents of municipal wastewater treatment plants varied from below 10 to 100 microg/L. The ranges of the concentrations in the Glatt River in ng/L were 636-3,690 for BT, 122-628 for TT, 68-326 for NP, 6-22 for OP and 9-76 for BPA. The corresponding mass flows in g/d were 93-1,870 for BT, 18-360 for TT, 24-183 for NP, 1-16 for OP and 2-72 for BPA. The concentrations and mass flows of NP in the River Glatt were drastically lower than the analogous values found 15 years ago. Thus, a substantially decreased environmental exposure can be observed due to the reduction of the use of alkyphenol polyethoxylate surfactants in Switzerland. The current concentrations of NP, OP and BPA are within the ranges reported for weakly impacted surface waters. The investigated contaminants occur at quantitatively measurable but varying concentrations in municipal wastewaters and in the Glatt River reflecting their ubiquitous input into wastewaters and their different behaviour during biological wastewater treatment.

Determination of thermodynamic parameters of ethoxylated nonionic surfactants by means of reversed-phase high-performance liquid chromatography

Abstract An approach to evaluate the thermodynamic properties of ethoxylated nonionic surfactants by using the reversed-phase high-performance liquid chromatography (RP-HPLC) is described. The enthalpies (Δ H °) and entropies (Δ S °) of transfer from the mobile to the stationary phase were determined using the change of capacity factor with temperature. The surfactants taken into consideration were homogeneous (C 10–16 E 8 ) and polydisperse (C 12–18 EO 10 ) ethoxylated fatty alcohols. The stationary phase was the alkyl-silica (i.e. octyl- and octadecyl-silica). The eluents were various methanol–water mixtures and the detection was done at 280 nm. The obtained Δ H ° and Δ S ° values were negative and decreased with the alkyl chain length of the surfactant, the water content of the eluent and the length of the ligand bonded to the stationary phase. The methylenic increments of enthalpy, entropy and Gibbs free energy of transfer from pure water as eluent to the nonpolar stationary phase were also determined. Their values were of −8.60 kJ mol −1 , −18.71 J mol −1 K −1 and −2.74 kJ mol −1 on the octyl-silica column and, respectively of −17.28 kJ mol −1 , −46.40 J mol −1 K −1 and −2.76 kJ mol −1 on the octadecyl-silica column. The enthalpy–entropy compensation behavior of the surfactants was evaluated and the compensation temperatures obtained were within the 455–586 K range. They agreed with those for alkylbenzenes and polycyclic aromatic hydrocarbons in the RPLC systems. The results reveal the likeness of the interactions involved in the retention of hydrophobic compounds on alkyl-bonded stationary phases and hydro-organic eluents.

Simultaneous determination of flucytosine and fluorouracil in human plasma by high-performance liquid chromatography.

A validated, sensitive and precise reversed-phase high-performance liquid chromatographic method for the simultaneous determination of 5-flucytosine (5-FC) and 5-fluorouracil (5-FU) in human plasma is described. Two compounds, 5-methylcytosine (5-MC) and 5-chlorouracil (5-CU), were used as internal standards for the determination of 5-FC and 5-FU, respectively. Plasma samples were deproteinized with trichloroacetic acid and chromatographed on an octylsilica column, maintained at 30 degrees C during elution, using a 0.04 M phosphate buffer, pH 7.0, as eleunt. Spectrophotometric diode array detection was used at 266 nm. 5-FC, 5-FU, 5-MC and 5-CU were found to have retention times of 4.8, 5.8, 7.7 and 11.0 min respectively. Recoveries of 91-120% with reproducibility and repeatability coefficients of variation of 0.8-6% were obtained. Mean correlation coefficients of 0.99989 and 0.9995 were found for the linear calibration curves (n = 2) of 5-FC (4.816-192.6 mg/l) and 5-FU (0.05368-5.368 mg/l), respectively. The limits of quantitation were 0.3 mg/l for 5-FC and 0.05 mg/l for 5-FU.

SEPARATION OF INTACT PHOSPHATIDYLCHOLINE MOLECULAR SPECIES BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Identification of the molecular species of phosphatidylcholines (PCs) is important for studies of lipid metabolism and the structure of cell membranes. The high performance liquid chromatography (HPLC) separations of the intact molecular species of PCs used earlier were mostly on reversed-phase C18 (octadecyl silica) columns, usually with isocratic elution. The eluent used contained a silanol suppressing agent. Volatile silanol suppressing agents are preferred for the purification of PCs because they can be removed from the HPLC fractions easily by nitrogen stream. They must be used when liquid chromatography-mass spectrometry (LC-MS) and evaporative light scattering detector (ELSD) are used because non-volatile silanol suppressing agents cause high background. For the identification of radioactive metabolites, using both the UV detector at 205 nm and radioactivity flow detector sequentially, we recommend the separation on a C8 (octyl silica) column, eluted with a gradient of methanol-water containing conc. NH4OH as silanol suppressing agent.

Chemiluminescent determination of cholesterol hydroperoxides in human erythrocyte membrane.

A method for separating, detecting, and quantifying cholesterol hydroperoxide (Ch-OOH) based on extraction, purification by solid-phase extraction cartridge, high-performance liquid chromatography with chemiluminescent detection (HPLC-CL), and liquid chromatography-mass spectrometry has been developed for human erythrocyte membrane. We prepared standard compounds of the cholesterol 5alpha-, 7alpha-, and 7beta-hydroperoxides (Ch 5alpha-OOH, Ch 7alpha-OOH, and Ch 7beta-OOH). An octyl silica column with methanol/water/acetonitrile 89:9:2 (by vol) as eluent was used to determine Ch-OOH. HPLC-CL that incorporated cytochrome c and luminol as the post-column luminescent reagent was used. We also investigated the optimal assay conditions and how to prevent formation of artifact Ch-OOH. Analysis of erythrocyte membranes from seven healthy volunteers identified Ch 7alpha-OOH and Ch 7beta-OOH, but not Ch 5alpha-OOH, as commonly occurring components. The respective mean concentrations of Ch 7alpha-OOH and Ch 7beta-OOH were 2.5+/-1.6 and 5.4+/-3.5 pmol/mL blood.

Peak distortion in reversed-phase liquid chromatography as a consequence of viscosity differences between sample solvent and mobile phase

Solute peaks may suffer severe distortions when their solutions in solvents whose viscosities differ from those of the mobile phase are injected in typical RPLC columns. A series of experiments with carefully chosen systems were performed, employing octyl silica columns, sample solvents whose viscosities were higher or lower than those of the corresponding mobile phases, and solutes that were more or less retained then the corresponding sample solvents. For a given viscosity difference, the importance of the distortions increases as the difference between solute and sample solvent retention times decreases. Chromatographic systems can be classified into four categories, and chromatograms illustrative of all of them were obtained.

Neural networks as a tool for modelling the retention behaviour of dihydropyridines in micellar liquid chromatography

Artificial neural networks have been applied for modelling the retention behaviour of a group of dihydropyridines in Micellar Liquid Chromatography and their capabilities have been compared with classical statistical methods (multiple regression). Some parameters concerning the architecture of the nets have been evaluated. Thus, type of transfer function in the hidden layer, number of neurons in the hidden layer and the use of recurrent networks and transformations of the output data have been optimized. These studies have been carried out with the retention data for 27 dihydropyridines in an octyl silica column using micellar mobile phases containing hexadecyl trimethylammonium bromide (CTAB) or sodium dodecyl sulphate (SDS) as surfactants and n-propanol or n-butanol as organic modifiers. From the results obtained some considerations can be drawn. The selection of the transfer function is very important to get good results, the logarithmoid function being the best one among the different functions studied. Also, the least errors were obtained when three nodes in the hidden layer and the reciprocal of retention factors (as the output variable) were employed.

Neural Network Capability for Retention Modeling in Micellar Liquid Chromatography with Hybrid Eluents

Until date no papers have been reported about modeling studies in Micellar Liquid Chromatography by means of neural networks, only classical statistical methods have been used. In this work an overview into the capabilities of neural networks for modeling retention data in Micellar Liquid Chromatography is presented. To solve the problem of capacity factor modeling some parameters have been evaluated: type of activation function, number of neurons in the hidden layer and the use of some input and/or output data transformations. These studies have been carried out with the retention data for twenty-three compounds (benzene derivatives and polycyclic aromatic compounds) in an octyl silica column using micellar mobile phases containing CTAB (hexadecyl-trimethylammonium bromide) as the surfactant and modified with n-propanol. From the results obtained some considerations can be drawn. The selection of the activation function is very important to get good results and the best ones have been obtained when a linear activation function, a recurrent network and a logarithm transformation (logarithm of the capacity factor) have been used.

Prediction and Modelling Studies for Capacity Factors of a Group of Dihydropyridines in Micellar Liquid Chromatography with Hybrid Eluents

Empirical equations were used to predict the retention behaviour of a group of twenty-seven dihydropyridines in Micellar Liquid Chromatography with hybrid eluents on an octylsilica column. A theoretical model was also used to study their retention mechanism. Hexadecyltrimethylammonium bromide and sodium dodecyl sulphate were used as surfactants in the mobile phase and as organic modifiers n-propanol and n-butanol were employed.

Influence of Alcohol Organic Modifiers Upon the Association Constants and Retention Mechanism for Aromatic Compounds in Micellar Liquid Chromatography

Abstract Solute-micelle association constants and partition coefficients between stationary, aqueous, and micellar phases have been determined for a group of benzene derivatives and polycyclic aromatic hydrocarbons with sodium dodecyl sulphate and hexadecyltrimethylammcnium bromide by Micellar Liquid Chromatography (MLC) with an octylsilica column using micellar mobile phases in absence and in the presence of organic modifiers (methanol, n-propanol, and n-butanol). The retention mechanism of these compounds in the chromatographic system has been studied by comparing experimental capacity factors and selectivity coefficients with those theoretically calculated assuming a direct transfer mechanism. When the surfactant concentration in the mobile phase is increased, a tendency to a change from a three-partition equilibrium mechanism to a direct transfer of solutes from micellar to stationary phase is observed for both surfactants. This change was favored when: (1) the hydrophobic character of the solute incr...

Direct plasma liquid chromatographic-tandem mass spectrometric analysis of granisetron and its 7-hydroxy metabolite utilizing internal surface reversed-phase guard columns and automated column switching devices

An alternative on-line automated sample enrichment technique useful for the direct determination of various drugs and their metabolites in plasma is described for rapid development of highly sensitive and selective liquid chromatographic methods using mass spectrometric detection. The method involves direct injection of plasma onto an internal surface reversed-phase (ISRP) guard column, washing the proteins from the column to waste with aqueous acetonitrile, and backflushing the analytes onto a reversed-phase octyl silica column using switching valves. The analytes were detected using a tandem mass spectrometer operated in selected reaction monitoring (SRM) mode using atmospheric pressure chemical ionization (APCI). Use of two ISRP guard columns in parallel configuration allowed alternate injections of plasma samples on these columns for sample enrichment and shortened the column equilibration and LCMSMS analysis times, thereby increasing the sample throughput. The total run time, including both sample enrichment and chromatography, was about 6 min. Using this technique, an analytical method was developed for the quantitation of granisetron and its active 7-hydroxy metabolite in dog plasma. Granisetron is a selective 5-HT3 receptor antagonist used in the prevention and treatment of cytostatic induced nausea and vomiting. Recovery of the analytes was quantitative and the method displayed excellent linearity over the concentration ranges tested. Results from a three day validation study for both compounds demonstrated excellent precision (1.3–8.7%) and accuracy (93–105%) across the calibration range of 0.1 to 50 ng/ml using an 80 μl plasma sample. The automated method described here was simple, reliable and economical. This on-line approach using ISRP columns and column switching with LCMSMS is applicable for the quantification of other pharmaceuticals in pharmacokinetics studies in animals and humans which require high sensitivity.

Simultaneous determination of granisetron and its 7-hydroxy metabolite in human plasma by reversed-phase high-performance liquid chromatography utilizing fluorescence and electrochemical detection

A highly sensitive and selective high-performance liquid chromatographic method was developed for the determination of granisetron and its active metabolite, 7-hydroxygranisetron (7OH-G) in human plasma. Granisetron is a selective 5-hydroxtryptamine receptor antagonist used in the treatment of cytotoxic drug-induced emesis. The method involves isolation of granisetron, 7OH-G and the internal standards from plasma by solid-phase extraction prior to reversed-phase ion-pair chromatographic separation on an octyl silica column with subsequent quantification of analytes simultaneously either with electrochemical (7OH-G) or fluorescence (granisetron) detectors which are placed in series. The recovery of granisetron and 7OH-G from human plasma was quantitative. Using 1 ml of plasma, the limits of quantification for granisetron and 7OH-G were 0.1 and 0.25 ng/ml, respectively. Linear responses in analyte/internal standard peak-area ratios were observed for analyte concentrations ranging from 0.1 to 50 ng/ml plasma. Precision and accuracy were within 13% across the calibration range for both granisetron and 7OH-G. The method was sufficiently sensitive, accurate and precise to support pharmacokinetic studies for granisetron and 7OH-G, in both normal and patient populations.

High-performance liquid chromatographic determination of the N-ethyl tricarbamate ester pro-drug of fenoldopam utilizing simultaneous post-column hydrolysis and fluorescence derivatization

A high-performance liquid chromatographic (HPLC) method was developed for the determination of SK&F 105058 (I), the N-ethyl tricarbamate ester pro-drug of fenoldopam, in dog plasma. Fenoldopam is a selective agonist of peripheral dopaminergic (DA-1) receptors and has been shown to improve blood flow and lower blood pressure. The method involves isolation of I and the internal standard (I.S.) from plasma by solid-phase extraction prior to chromatographic separation on an octyl silica column. Following chromatographic separation, the carbamate esters of I and I.S. were simultaneously hydrolyzed and the liberated alkylamines were derivatized, by mixing the column effluent with an alkaline solution of o-phthaldialdehyde and a thiol, to generate a highly fluorescent isoindole product which was subsequently detected with a fluorometer. Optimization of chromatographic and post-column reaction conditions resulted in an on-column detection limit of 0.4 ng. The recoveries for I and I.S. from plasma were 80.0 ± 5.0% and 62.0 ± 4.0%, respectively. The limit of quantification for I using 0.2 ml of plasma was 5 ng/ml. Linear response was observed for concentrations of I ranging from 5 to 2000 ng/ml plasma. The method was suitably specific and sensitive for pharmacokinetic and metabolic studies of I in dogs. The methodology developed should be generally applicable to the determination of carbamate-type pro-drugs in biological media.

Determination of a novel hematoregulatory peptide in dog plasma by reversed-phase high-performance liquid chromatography and an amine-selective o-phthaldialdehydethiol post-column reaction with fluorescence detection

A sensitive and selective high-performance liquid chromatographic method was developed for the determination of SB 107647 ( I), a novel synthetic hematoregulatory peptide, in plasma samples of dog and rat. The method involves isolation of I and the internal standard (SB 203285, IS) from plasma by a solid-phase anion-exchange extraction column prior to reversed-phase ion-pair chromatographic separation on an octyl silica column. Following separation, a selective post-column reaction of the ϵ-amino groups of the lysine moieties of the peptide with o-phthaldialdehyde and a thiol under basic conditions was used to generate a highly fluorescent isoindole product, which was subsequently detected on-line with a fluorometer. Optimization of chromatographic conditions resulted in an on-column detection limit of 1 ng. The recovery of I from dog plasma at 20 and 4000 ng/ml was 50.0 ± 5.94 and 56.6 ± 1.45% (Mean ± S.D.), respectively. The limit of quantification for I, for 0.25-mi plasma samples, was 20 ng/ml. Linear response was observed for concentrations of I ranging from 20 to 4000 ng/ml of plasma. The assay was sufficiently sensitive, accurate and precise to support toxicokinetic studies in animal species.