High-performance liquid chromatography (HPLC) determination of inosine, a potential biomarker for initial cardiac ischaemia, using isolated mouse hearts.

Each year in the USA approximately 7-8 million patients with non-traumatic chest pain come to hospital emergency rooms. It is estimated that approximately 2-5% of these patients are experiencing cardiac ischaemia, but due to the shortcomings of the available testing methods they are incorrectly diagnosed and discharged without appropriate therapy having been provided. Preliminary data with a globally ischaemic mouse heart model has demonstrated that endogenous inosine might be a potential biomarker of initial cardiac ischaemia before cardiac tissue necrosis. A high-performance liquid chromatographic diode array detection (HPLC-DAD) method was utilized for the detection and quantification of inosine in Krebs-Henseleit (Krebs) buffer solution perfusing from surgically removed and isolated mouse hearts undergoing global cardiac ischaemia. A C18 column at a flow rate of 0.6 ml min-1 with an aqueous mobile phase of trifluoroacetic acid (0.05% trifluoroacetic acid in deionized water, pH 2.2, v/v) and methanol gradient was used for component separation. The assay detection limit for inosine in Krebs buffer solution was 500 ng ml-1 using a 100-microl neat injection. The HPLC results were used to determine total cardiac effluxed inosine into the Krebs effluent for each mouse during oxidative stress and compared with the per cent cardiac ventricular functional recovery rate to determine if a relationship exists amongst this cardiovascular parameter during periods of cardiac oxidative stress.

The effect of alkylamine additives on the sensitivity of detection for paclitaxel and docetaxel and analysis in plasma of paclitaxel by liquid chromatography-tandem mass spectrometry.

The formation of multiple molecular ions, especially due to sodium adduct ion formation, is commonly observed in electrospray mass spectrometry and may make reproducible and sensitive quantitation difficult. The objective of this work was to investigate the underlying mechanism involved in the suppression of multiple molecular ion formation and to improve the sensitivity of detection for the two anti-neoplastic agents paclitaxel and docetaxel. The results showed that alkylamine additives could significantly improve the detection of paclitaxel and docetaxel by suppression of multiple molecular ions through preferential formation of a predominant alkylamine adduct ion. Possible binding sites, binding interactions and binding competition were investigated for the sodium adduct and alkylamine adduct ions using various experimental techniques. The formation of a predominant amine adduct ion may be due to increased surface activity in the droplet. The optimal alkylamine for both analytes was octylamine, which increased peak heights of paclitaxel and docetaxel 4.8 and 3.7-fold (n = 3), respectively. The precision of the signals for the analytes was also improved 5.7-fold. A quantitative assay in plasma for paclitaxel was partially validated for the calibration range 1.0-1000 ng/mL (r = 0.9977) when using 0.05% octylamine as a reconstitution solution additive. The limit of detection (LOD) and limit of quantitation (LOQ) were 0.5 and 0.9 ng/mL, respectively. Acceptable precision, accuracy, specificity and sample stability were demonstrated for this assay. This approach may prove useful for other analytes with similar binding sites.

Sensitivity enhancement in liquid chromatography/atmospheric pressure ionization mass spectrometry using derivatization and mobile phase additives.

High performance liquid chromatography with atmospheric pressure ionization (API) mass spectrometry has been essential to a large number of quantitative analytical applications for a variety of compounds. Poor detection sensitivity however is a problem observed for a number of analytes because detection sensitivity can be affected by many factors. The two most critical factors are the chemical and physical properties of the analyte and the composition of the mobile phase. In order to address these critical factors which may lead to poor sensitivity, either the structure of the analyte must be modified or the mobile phase composition optimized. The introduction of permanently charged moieties or readily ionized species may dramatically improve the ionization efficiency for electrospray ionization (ESI), and thus the sensitivity of detection. Detection sensitivity may also be enhanced via introduction of moieties with high proton affinity or electron affinity. Mobile phase component modification is an alternative way to enhance sensitivity by changing the form of the analytes in solution thereby improving ionization efficiency. pH adjustment and adduct formation have been commonly used to optimize detection conditions. The sensitivity of detection for analytes in bio-matrices could also be enhanced by decreasing ion-suppression from the matrix through derivatization or mobile phase addition. In this review, we will discuss detection-oriented derivatization as well as the application of mobile phase additives to enhance the sensitivity of detection in liquid chromatograph/atmospheric ionization/mass spectrometry (LC/API/MS), focusing in particular on the applications involving small molecules in bio-matrices.

A novel incubation direct injection LC/MS/MS technique for in vitro drug metabolism screening studies involving the CYP 2D6 and the CYP 3A4 isozymes.

A direct injection LC/MS/MS method involving a novel incubation technique was developed for the inhibition screening of CYP 2D6 and CYP 3A4 isoenzymes using dextromethorphan and midazolam as probe substrates. Both assays were performed using an electrospray ionization source in the positive ion mode. Direct injection was possible by using a short C 18, LC column (2 mm x 20 mm) with large particle diameter packing (10 microm). Analytical characteristics of the direct injection technique were studied by examining matrix effects, which showed suppression of the ESI signal between 0.20 and 0.65 min. The retention times for analytes were adjusted to approximately 0.8 min (k'>3), resulting in no matrix effect. Column lifetime was evaluated and determined to be approximately 160 direct injections of the matrix. The precision and accuracy of the control samples for the quantitation of dextromethorphan was between -0.53 and -12.80, and 3.73 and 6.69% respectively. Unlike conventional incubation techniques, incubations were carried out in an autosampler equipped with a heating accessory. This novel incubation method, which involved no stirring of the incubation mixture, estimated the Cl(int in vitro) for dextromethorphan and midazolam in human liver microsomes to be 1.65+/-0.22 ml/(hmg) and 0.861 ml/(min mg) respectively. The autosampler tray maintained uniform temperature and was sensitive to changes in temperature between 33 and 41 degrees C. High-throughput screening was performed using known inhibitors of the CYP 2D6 isozyme, and the system was evaluated for its ability to differentiate between these inhibitors. The strong inhibitor quinidine resulted in a 25.6% increase in t(1/2), the medium potency inhibitor chlorpromazine resulted in an increase of 6.14% and the weak inhibitor primaquine had no significant effect on half-life. This technique involves no sample preparation, demonstrated run times of 2 min per injection and can be fully automated. The method should therefore prove to be a valuable tool in the drug discovery process.

A study of matrix effects on an LC/MS/MS assay for olanzapine and desmethyl olanzapine.

The purpose of this research project was to investigate potential matrix effects of anticoagulant and lipemia on the response of olanzapine, desmethyl olanzapine, olanzapine-D(3) and desmethyl olanzapine-D(8) in an LC/MS/MS assay. Blank human serum and sodium heparin, sodium citrate, and K(3)EDTA plasma with various degrees of lipemia were fortified with olanzapine, desmethyl olanzapine, olanzapine-D(3) and desmethyl olanzapine-D(8). Six replicates of each sample were extracted using Waters Oasis MCX cartridges and analyzed using electrospray LC/MS/MS. The analytes were separated on a Phenomenex LUNA phenyl hexyl, 2 mm x 50 mm, 5 microm, analytical column and a gradient rising from 2 to 85% mobile phase B. Mobile phase A consisted of acetonitrile-ammonium acetate (20 mM) (52:48 v/v) and mobile phase B was formic acid-acetonitrile (0.1:100 v/v). Ion suppression was investigated through post column infusion experiments. The degree of lipemia of each sample, indicated by turbidity, was ranked into categories from least to greatest and used for statistical analyses. The results from analysis of variance testing indicated that lipemia, anticoagulant and their interaction significantly influenced mass spectral matrix effects and extraction matrix effects. Differential behavior between the analytes and labeled internal standards contributed to variability. The most significant source of variability however, was ion suppression due to co-eluting matrix components.

Characterization of quinidine 3-hydroxylation as a probe for the CYP 3A enzyme using a novel capillary electrophoresis technique.

Capillary electrophoresis (CE) with a direct injection technique was used to characterize the formation of (3S)-3-hydroxyquinidine (3-OHQ) as a probe for the CYP 3A isoenzymes in rat liver microsomes. Detection was performed either in the absorbance mode or by employing laser-induced fluorescence (LIF) detection. Michaelis-Menten parameters (mean values+/-S.D.) K(m) and V(max) for the formation of 3-OHQ from the probe drug quinidine sulfate (QS) in rat liver microsomes were 37+/-4.6 micro g/ml (47.1+/-5.9 micro M) and 321+/-4 ng/mg/h (942+/-11.7 pmol/mg/h), respectively. Inhibition studies were performed to evaluate the specificity of 3-OHQ as a probe for the CYP 3A enzyme. Ketoconazole and fluconazole were found to be inhibitors of 3-OHQ formation and exhibited K(i) values of 0.19 and 20.1 micro M, respectively. Inhibition with the weak inhibitor, erythromycin could only be estimated using LIF detection due to lack of sensitivity in the absorbance mode. The formation of 3-OHQ in rat liver microsomes can be used as a model for the screening of the CYP 3A enzyme. Direct injection, ensures faster analysis time due to the lack of sample preparation and the low volume capabilities of the technique makes it attractive for the screening of a large number of compounds.

Determination of (3S)-3-hydroxy quinidine for metabolism screening experiments using direct injection capillary electrophoresis and laser-induced fluorescence detection.

Capillary electrophoresis (CE) has been used with collinear laser-induced fluorescence detection (LIF) to determine the amount of (3S)-3-hydroxy quinidine (3OHQ) formed on direct injection of microsomal incubation mixtures. 3OHQ is the CYP 3A4 metabolite of quinidine sulfate (QS) and is therefore useful for metabolism screening studies. The method was validated analytically and tested for its capability of screening for a weak inhibitor of the CYP 3A4 isozyme. A linear calibration was found to provide the best fit for the standard curve with a correlation of 0.9950 and all concentration residuals less than 15%. The percentage relative standard deviations (RSDs) of two controls, 175 and 2250 ng/mL, were 9.29 and 5.68% and the percentage differences from normal (DFN) were 6.87 and -4.37%, respectively. The concentration limit of detection (LOD) for 3OHQ in the incubation matrix was 52.11ng/mL and the mass LOD was approximately 521.1 fg (injection volume 10 nL). The effectiveness of the method to screen for the weak inhibitor erythromycin has been shown by calculating percentage inhibition when incubating with different concentrations of QS. Sensitive detection coupled with the convenience of the direct injection technique makes this an attractive approach for metabolism screening. The small sample size capability of CE will further reduce the quantities of probe drug, microsomes and other reagents required for incubation studies.

Determination of amphetamine in dog plasma by gas chromatography with mass selective detection.

This paper describes the validation of an analytical method for the determination of amphetamine in beagle dog plasma by gas chromatography coupled to mass spectrometry (GC-MS). d-Amphetamine-d(6) was used as the internal standard. The method consisted of a rapid single-step liquid-liquid extraction and derivatization of amphetamine with 2,2,2-trichloroethyl chloroformate, followed by sensitive GC-MS detection. This is the first report utilizing the combination of trichloroethyl chloroformate as a derivatization reagent and a deuterated amphetamine analog as an IS for the quantification of amphetamine in plasma. The method was validated in terms of specificity, curve fit, precision, accuracy, recovery and stability, and was acceptable according to FDA draft guidelines for validation of bioanalytical methods. The limit of detection was 0.65 ng/mL. The calibration range was 5-150 ng/mL. The validated method was successfully employed for the quantitation of amphetamine in dog plasma samples for pharmacokinetic profiling.

A cellular automata model of chromatography.

Dynamic models of the behavior of solvent and solute molecules can be made using cellular automata. A chromatographic column was represented by use of a cellular automata grid of 43 x 200 spaces. Solvent (mobile phase), solute and stationary phase cells were designated to simulate the chromatographic situation. The movements of solute and solvent cells down the grid were monitored for different numbers of iterations, different flow rates and different affinities of the solutes for the stationary phase and the solvent for itself. The cellular automata dynamics were successfully able to model expected chromatographic behavior except in a few cases where the number of cells was not large enough to provide an average value reflective of the molecular situation.

High-performance liquid chromatography with on-line post-column immunoreaction detection of digoxin and its metabolites based on fluorescence energy transfer in the far-red spectral region.

The combination of immunoassays with separation techniques such as chromatography can result in enhanced selectivity and sensitivity. This paper describes an on-line chromatography with immunochemical post-column fluorescence energy transfer detection for digoxin and its metabolites. R-phycoerythrin (PE) was used as the donor and an indodicarbocyanine dye (Cy5) as the acceptor label. These labels allow the detection in the far-red spectral region, which is more selective for biological samples. Hence, digoxin was labeled with PE using the activated digoxigenin-NHS-ester and monoclonal anti-digoxin antibody was labeled with Cy5. Digoxin and its metabolites was injected into the HPLC system followed by post-column injection of R-phycoerythrin labeled digoxin and by Cy5 labeled anti-digoxin antibody. Incubation time was provided using an open tubular reactor coil at room temperature. The detection was performed by measurement of the sensitized emission of Cy5 at 670 nm due to fluorescence energy transfer from PE labeled with digoxin. The system was optimized with regard to the concentrations of the used post-column reagents as well as incubation time and temperature. The dynamic range of digoxin spiked in 0.01 M phosphate buffer (pH 7.4) was 0.05 to 10 ng/ml with a correlation coefficient of 0.989. The limit of detection was 33 pg/ml. The precision of two controls, 0.4 and 4 ng/ml, was found to be 2.2 and 8.7% RSD, respectively, accuracy was 10.7 and 20.3% (n=6 in each case).

Coupling immunoassays with chromatographic separation techniques.

Coupling immunoassays with HPLC separation techniques is becoming increasingly useful in the analysis of biological and nonbiological samples of both large and small molecules. This is because it provides both sensitivity and selectivity for molecular analysis at relatively low cost, low maintenance and with excellent potential for automation. This paper reviews application of this hyphenated approach both in the pre-column immunoextraction and post-column immunodetection modes. Systems in which immunoassays are interfaced to chromatographic separations in order to separate bound and free fractions of the immunoassay will not be included since these systems do not provide the enhanced selectivity common to hyphenated systems. Post-column immunodetection is based on various immunoassay formats such as direct detection, one-site, competitive and sandwich immunoassays. Homogeneous immunodetectors are more convenient than heterogeneous immunodectors since there are no separation and column regeneration steps involved in homogeneous immunoassays. On the other hand, heterogeneous immunoassays are generally more sensitive than homogeneous immunoassays since interfering substances are removed prior to immunodetection. Advantages and limitations for the various approaches will be discussed.

Post-column reaction detection of biotin in human plasma ultrafiltrate based on laser-induced fluorescence energy transfer in the far-red spectral region.

High performance liquid chromatography followed by post-column reaction detection in the far-red spectral region provides added sensitivity and selectivity. A homogeneous fluorescence energy transfer assay in the competitive mode based on the binding of biotin and streptavidin was developed as an on-line post-column reaction detection system. The labels used for energy transfer were R-Phycoerythrin conjugated to biotin and Cyanine 5 labeled with streptavidin. The energy transfer peak was measured at 670 nm and excitation was achieved using the 488 nm line of an argon ion laser. The biotin concentration in plasma ultrafiltrate ranged from 0.024 to 6.12 ng/mL (n = 6). The precision of the two controls, 0.24 and 2. 44 ng/mL, was found to be 18.70% and 9.92% relative standard deviation respectively. Accuracy was 10.47% and 1.95% difference from spiked, respectively (n = 6). The limit of detection was 21.70 pg/mL (8.90 x 10(-11)M) calculated based on a factor of 2x the standard deviation of the blank (n = 6). The correlation coefficient for the calibration curve was found to be 0.9995. Recovery from plasma ultrafiltrate at 2.44 ng/mL was 103.40% (n = 6). Detection selectivity was indicated by the absence of background fluorescence in six different plasma samples collected from six individual donors. Endogenous levels were detected in two of the six pools of plasma ultrafiltrates.

Visible diode laser induced fluorescence detection of doxorubicin in plasma using pressurized capillary electrochromatography.

Pressurized capillary electrochromatography is a variant of capillary electrochromatography (CEC) in which the driving force is both electroosmotic and hydraulic. The inlet of the CEC capillary is pressurized using an HPLC pump, and an electric field is simultaneously applied. This work describes a method for the analysis of doxorubicin. Doxorubicin was reacted with Cy5.29.OSu in acetonitrile. The derivative was confirmed by RP-TLC. A CEC system equipped with a VDLIF detector was constructed and used to analyze the derivative. The reaction mixture was injected onto a capillary packed in-house with 3 microm C-18 Luna particles and separation was carried out at 25 kV using 70% acetonitrile/ 30% phosphate (10 mM, pH = 4.8) as the mobile phase. The derivatization reaction was optimized by the investigation of parameters such as reaction time, temperature and concentration of label in order to increase the yield of the derivative. The optimal conditions were determined to be 30 min, 80 degrees C and 50 nmol/mL, respectively. Doxorubicin was extracted from plasma using solid-phase extraction under alkaline conditions, derivatized and injected onto the CEC-VDLIF system. The selectivity of the assay was demonstrated by a lack of interfering peaks due to plasma constituents across the elution window of the derivative peak in blank plasma extracts (n = 6 sources). The limit of detection (LOD) of the assay in plasma calculated as 3 s(b)/m was determined to be 1.7 ng/mL. The precision of the assay determined at a concentration of 167.7 ng/mL (n = 5) was found to be within 7.04 %RSD.

Post-column reaction detection based on fluorescence energy transfer in the far red spectral region.

Post-column reaction detection may result in enhanced analytical sensitivity and selectivity. This paper describes an on-line HPLC with post-column fluorescence energy transfer assay using biotin as a model analyte. Biotin labeled with R-phycoerythrin was used as the donor labeled ligand and streptavidin labeled with an indodicarbocyanine dye (Cy5), the acceptor labeled binder protein. The use of these labels provided a detection wavelength in the far red spectral region which is more selective for biological samples. In the on-line system, biotin was injected into the HPLC system followed by Cy5 labeled streptavidin and R-phycoerythrin labeled biotin, post-column. The mixture was incubated on-line in an open tubular reactor coil maintained at 37 degrees C. The measured response was the sensitized emission of Cy5 due to fluorescence energy transfer from R-phycoerythrin labeled biotin measured at 670 nm. Excitation was at 488 nm, which provided a large Stokes shift for reduction of scatter interference. The system was optimized with regard to the post-column reagents to obtain the minimum detectable concentration while maintaining appropriate dynamic range for the analysis of biotin. Biotin spiked in 0.01 M phosphate buffer, pH 7.4, showed a dynamic range of 304.0 pg/ml-122.20 ng/ml with a correlation coefficient of 0.993. The limit of detection for this assay was 304.0 pg/ml. The precision calculated at the blank (n = 6) was 4.14%.

Pharmacokinetics of minoxidil in patients with cirrhosis and healthy volunteers.

OBJECTIVES: To determine the effect of reduced hepatic function on the pharmacokinetics of minoxidil. The pharmacokinetics of antipyrine, lorazepam, and indocyanine green were included as indicators of hepatic function. METHODS: Eight mild cirrhotics and eight healthy subjects received antipyrine (po), lorazepam (IV), indocyanine green (IV) and minoxidil 5 mg (po). Blood and urine were sampled for up to 72 h after each drug, and drug concentrations were measured by validated HPLC methods. Blood pressure and heart rate were measured for safety. RESULTS: For unchanged minoxidil, the serum elimination rate constant was significantly smaller and mean residence time was significantly longer in cirrhotic patients. Urinary elimination rate constant for minoxidil glucuronide was significantly smaller and fraction of dose excreted in urine was significantly higher in cirrhotic patients. Antipyrine elimination was significantly slower for cirrhotic patients. No differences were observed in lorazepam pharmacokinetic parameters. CONCLUSION: Pharmacokinetic analysis suggests a longer dosage interval may be appropriate in patients with hepatic impairment. In the absence of multiple-dose minoxidil pharmacodynamic studies in this population, minoxidil should be used as in other populations: begin with a modest dose, and adjust the dose based on clinical response.

Investigation of far red dyes for use in peroxyoxalate chemiluminescence detection and analysis of the cy5 derivative of amantadine hydrochloride in human plasma.

Peroxyloxalate chemiluminescence is well established as a tool for improvement of selectivity and sensitivity for chemiluminophores and their derivations in HPLC eluates. Chemiluminescence in the far-red spectral region was investigated in this work to further enhance the sensitivity of chemiluminescence through more efficient singlet excitation energy transfer and to enhance the selectivity of the approach through a reduction in matrix and scatter interference. A number of fluorescent compounds that can be excited in the UV, visible and far red spectral regions were investigated for chemiluminescence yield using the bis(2,4,6-trichlorphenyl) oxylate reaction. It was found that a trend of increasing chemiluminescence with increasing excitation wavelength could be observed with cy5 providing the most efficient chemiluminescence. The succinate ester of cy5 was used to derivatize amantadine hydrochloride, an antiparkinsons drug, to form the derivative. The derivative was separated from reaction by products by C18 reversed phase HPLC and detected using a Soma S-3400 chemiluminescence detector. The detection limit for the diluted derivative was 200 femtomoles on column and sufficient for plasma analysis. Selectivity in plasma was demonstrated through derivatization of extracts of plasma that had been spiked with amantadine hydrochloride.

Visible diode laser induced fluorescence detection for capillary electrophoretic analysis of amantadine in human plasma following precolumn derivatization with Cy5.29.OSu.

Visible diode laser induced fluorescence (VDLIF) detection (620-700 nm) has become important in bioanalysis due to the increased sensitivity and selectivity that can be achieved in biological matrices. A selective and sensitive capillary electrophoretic method employing VDLIF detection has been developed for the analysis of amantadine in plasma. Amantadine was extracted from plasma into toluene under alkaline conditions and the residue was derivatized with the far-red label Cy5.29.OSu. The reaction mixture was dried under nitrogen, reconstituted and then injected onto a laboratory constructed capillary electrophoresis system equipped with a laboratory constructed visible diode laser detector temperature tuned to oscillate at 647.8 nm. The selectivity of the technique was evaluated by demonstrating a lack of interfering peaks in extracts of blank plasma. A calibration curve ranging from 1.8 to 461.1 ng ml(-1) was shown to be linear. The precision and accuracy of the assay (n = 6) were determined to be within 17% R.S.D. and 15% difference from the nominal concentration respectively. The limits of detection for unextracted amantadine and for amantadine from the extracted concentrate from plasma were determined to be 9.5 fmol and 115 amol respectively.

Visible diode laser-induced fluorescence detection of phenylacetic acid in plasma derivatized with Nile blue and using precolumn phase transfer catalysis.

This study reports the application of Nile blue (NB), a farred oxazine label, as a precolumn derivatization reagent for the measurement of free levels of phenylacetic acid (PAA) in plasma. The measurement of PAA in psychiatric populations is important because it provides a marker for 2-phenylethylamine (PEA), which has been implicated in the pathogenesis of schizophrenia and major depression. PAA was derivatized with NB through an amide linkage in the presence of 2-chloro-1-methylpyridinium iodide (carboxylic acid activator, CMP) and triethylamine (base catalyst, TEA), respectively. The formation of the NB-PAA derivative was confirmed using normal phase and reversed phase thin-layer chromatography, reversed phase liquid chromatography, and electrospray mass spectrometry. The formation of the NB-PAA derivative was optimized using a sequential single factor approach. The optimal conditions for the formation and chromatographic separation of the derivative were determined to be 8.0 nmol/mL NB, 390 nmol/mL CMP, 2 mumol/mL TEA, a reaction time of 45 min, and a reaction temperature of 25 degrees C. This derivatization scheme was performed in a phase transfer catalysis mode that enabled the simultaneous extraction, preconcentration, and derivatization of the analyte in a single step. The limit of derivatization of PAA was determined to be 1.0 x 10(-9) M in phosphate-buffered saline, a PAA-free matrix. This derivatization was limited not by the kinetics of the reaction but by the chromatographic separation of the derivative from a side reaction product. The method was used to estimate endogenous free levels of PAA in human plasma samples. The levels of PAA in four sources of plasma were determined to be within 30-70 ng/mL using the method of standard addition and reflected levels that have been reported in the literature. The limit of detection of the derivative was determined to be 7.33 x 10(-11) M using a laboratory-constructed HPLC-VDLIF detector.