Determination of nefazodone and its pharmacologically active metabolites in human blood plasma and breast milk by high-performance liquid chromatography

A method is described for the determination of nefazodone and its active metabolites hydroxynefazodone, the dione BMS-180492 and m-chlorophenylpiperazine in blood plasma and expressed human milk based on reversed-phase high-performance liquid chromatography. Measurements were performed on drug-free plasma and expressed human milk spiked with nefazodone and metabolites to prepare and validate standard curves and specimens collected from nursing mothers. Parent drug and metabolites were separated from the biological matrices by solid-phase extraction using CERTIFY columns. Chromatographic separation was achieved with a C₁₈ column and compounds were detected by their absorbance at 205 nm. Trazodone was used as an internal standard. The assay was validated for each analyte in the concentration range 200 to 1200 ng/ml.     

Column-switching high-performance liquid chromatographic detection of pholcodine and its metabolites in urine with fluorescence and electrochemical detection

A sensitive and selective method for the detection of pholcodine and its metabolite morphine in urine using high-performance liquid chromatography is described. It involves on-line clean-up of urine on a trace enrichment column packed with a polymeric strong cation-exchange material. Pholcodine and its metabolites were separated on two analytical columns with different selectivities. Pholcodine was detected by a fluorescence detector and morphine was detected electrochemically. One system, based on reversed-phase chromatography, applied a polystyrene—divinylbenzene column and gradient elution. The other system was based on normal-phase chromatography with a silica column and isocratic elution. Morphine was confirmed to be a metabolite of pholcodine by reversed-phase chromatography and electrochemical detection. Two unidentified metabolites of pholcodine were separated from pholcodine by normal-phase chromatography and detected by fluorescence detection.     

Simultaneous determination of the peptide-mitomycin KW-2149 and its metabolites in plasma by high-performance liquid chromatography

A gradient high-performance liquid chromatographic (HPLC) method is described for the quantification of KW-2149 and its two major metabolites in plasma. The method involves a sample clean-up by solid-phase extraction on C₁₈ columns, separation of the respective compounds by HPLC on a YMC ODS-AQ column (5-μm particle size, 150×6 mm I.D.), using a methanol–water gradient system as an eluent, and measurement by UV absorbance detection at 375 nm. The limits of quantitation were 10 ng/ml for KW-2149 and M-16, and 15 ng/ml for M-18. Recoveries from plasma were higher than 92% on C₁₈ extraction columns. Intra-day precision, expressed as %C.V., was between 1.4 and 6.5%. Intra-day accuracy ranged from 94 to 107%. Precision and accuracy of variability of inter-assays increased somewhat; however, were still within acceptable ranges. The ability of the method to quantify KW-2149 and two major metabolites simultaneously, with precision, accuracy and sensitivity, make it useful in monitoring the fate of this new mitomycin in cancer patients.     

Simultaneous extraction and separation of liquiritin,glycyrrhizic acid,and glabridin from licorice root with analytical and preparative chromatography

Simultaneous extraction and separation of liquiritin, glycyrrhizic acid, and glabridin from licorice were developed by liquidliquid extraction with liquid chromatography separation. By utilizing different extraction solvents, procedures, and times, the optimum extraction conditions were established. The extracts of licorice were separated and determined using a C₁₈ column with a mobile phase consisting of acetonitrile-water (containing 1.0% acetic acid) with a gradient elution of 0∼10 min from 20:80 to 60:40 (v/v). Preparative columns with different packing sizes were investigated to isolate the three compounds from the extracts of licorice. The 12 μm chromatographic column showed better separation for the three compounds from licorice. 0.29 mg/g for liquiritin, 1.43 mg/g for glycyrrhizic acid, and 0.07 mg/g for glabridin were obtained and the recoveries were 80.8, 89.7, and 72.5%, respectively.     

Determination of serotonin,its precursors,metabolites and [3H]serotonin in lung by high-performance liquid chromatography with fluorescence detection

A rapid, simple, sensitive method for the determination of serotonin, its precursors, metabolites and [³H]serotonin in lung is described. Tissue preparation requires only homogenization in dilute perchloric acid and centrifugation prior to separation by high-performance liquid chromatography using a reversed-phase column. Detection is based on the native fluorescence of indole compounds and detection limits ranged between 30–90 pg injected. The method has been used to determine these compounds in mouse lung and plasma.     

High-performance liquid chromatographic separation and isolation of quinidine and quinine metabolites in rat urine

A procedure for the separation and isolation of the urinary metabolites of quinidine and quinine by reversed-phase high-performance liquid chromatography is described. Nine metabolites of quinidine and eight metabolites of quinine were detected in the urine of male Sprague-Dawley rats after a single dose of quinidine or quinine (50 mg kg^?1). Following extraction from urine, the metabolites were separated on either an analytical or a semi-preparative reversed-phase column by gradient elution. After isolation and derivatization, the metabolites were analyzed by gas chromatography and gas chromatography—mass spectrometry.     

Application of microbore HPLC in combination with tandem MS for the quantification of rosuvastatin in human plasma

The potential of microbore high-performance liquid chromatography (HPLC) in combination with tandem mass spectrometry (MS/MS) for the sensitive detection of rosuvastatin (Crestor) in human plasma was investigated. Three microbore HPLC columns with internal diameters (i.d.) of 0.5, 1.0 and 2.0 mm were evaluated for column efficiency and mass sensitivity, and compared to a conventional 4.6 mm i.d. column. The 2.0 and 1.0 mm i.d. columns performed very well while the 0.5 mm i.d. column was slightly less efficient, this is probably due to a lower packing density. Good results with respect to gains in mass sensitivity compared to the conventional analytical column were achieved with the 2.0 and 1.0 mm columns. Thus, the 2.0 mm i.d. column had an improved signal-to-noise (S/N) ratio of 16 whilst the 1.0 mm i.d. column had an improved S/N ratio of greater than 70. Experiments with the 1.0 mm i.d. HPLC column were performed to determine the robustness of the microbore method for human plasma extracts after sample preparation using solid-phase extraction (SPE). A number of problems were encountered with extracts including high backgrounds, the blocking of the column and a rapid deterioration in column performance. The blocking of the column by particulates was solved by off-line filtration of the sample extracts. Peak tailing of the analytes and high background, both of which were due to endogenous interferences in the extracts, were eliminated using gradient elution. Using these approaches over 500 injections of plasma extracts were achieved without significant deterioration in assay performance. Quantities of rosuvastatin of 0.3 pg on-column could be detected and cross-validation experiments demonstrated that the conventional and the microbore HPLC-MS/MS methods provided similar information on the concentration of rosuvastatin but with greatly reduced sample consumption using the microbore method.     

Fluorometric determination of -fenfluramine, -norfenfluramine and phentermine in plasma by achiral and chiral high-performance liquid chromatography

High-performance liquid chromatography (HPLC) with fluorescence detection has been developed for the simultaneous determination of sympathomimetic amines including ephedrine, norephedrine, 2-phenylethylamine, 4-bromo-2,5-dimethoxyphenylethylamine, phentermine (Phen) and -fenfluramine (Fen) in spiked human plasma. Furthermore, an enantioselective HPLC method for the separation of -Fen (dexfenfluramine) and -Fen (levofenfluramine) in addition to their active metabolites - and -norfenfluramine (Norf) is described. The detection was achieved at emission wavelength of 430 nm with excitation wavelength of 325 nm for both methods. The analytes were extracted from plasma (100 μl) at pH 10.6 with ethyl acetate using fluoxetine as the internal standard. The extracts were evaporated and derivatized with the fluorescence reagent 4-(4,5-diphenyl-1H-imidazole-2-yl)benzoyl chloride in the presence of carbonate buffer (pH 9.0). A gradient separation was achieved on a C₁₈ column for the achiral separation or on a Chiralcel OD-R column for the chiral separation. The methods were fully validated, and shown to have excellent linearity, sensitivity and precision. The chiral method has been applied for the determination of - and -enantiomers of Fen and Norf, in addition to Phen in rat plasma after an intraperitoneal administration of -Fen and Phen, simultaneously.     

Evaluation of fused-core and monolithic versus porous silica-based C18 columns and porous graphitic carbon for ion-pairing liquid chromatography analysis of catecholamines and related compounds

This paper evaluates the performances of reversed-phase (RPLC) and ion-pairing chromatography (IPLC) coupled with UV detection for the analysis of a set of 12 catecholamines and related compounds. Different chromatographic columns (porous C18-silica, perfluorinated C18-silica, porous graphitic carbon, monolithic and fused-core silica-based C18 columns) were tested using semi-long perfluorinated carboxylic acids as volatile ion-pairing reagents. Much more promising results were obtained by IPLC than by RPLC and important improvements in analytes peak symmetry and separation resolution were observed when using the "fast chromatography" columns (monolithic and fused-core C18) under IPLC conditions. For UV detection, a satisfactory separation of the 12 selected analytes was achieved in less than 20 min by using a fused-core particles column (Halo C18) and a mobile phase composed of a 1.25 mM nonafluoropentanoic acid aqueous solution and methanol under gradient elution mode. The chromatographic method developed can be directly coupled with electrospray ionization tandem mass spectrometry (ESI-MS/MS) in positive ionization mode and 10 solutes among those selected can be observed. The presence of the acidic ion-pairing reagent in the mobile phase makes this system incompatible with negative ionization mode and thus unable to detect the two acidic compounds that only responded in negative mode. In terms of MS detection, Monolithic C18 column proved to be the best one to reach the lowest detection limits (LODs) (from 0.5 ngmL(-1) to 10 ngmL(-1) depending on the neurotransmitter). The applicability of the optimized LC-MS/MS method to a "real world" sample was finally evaluated. The presence of the matrix leads to signal suppression for several solutes and thus to higher LODs.     

Quantitation of promethazine and metabolites in urine samples using on-line solid-phase extraction and column-switching

A chromatographic method for the quantitation of promethazine (PMZ) and its three metabolites in urine employing on-line solid-phase extraction and column-switching has been developed. The column-switching system described here uses an extraction column for the purification of PMZ and its metabolites from a urine matrix. The extraneous matrix interference was removed by flushing the extraction column with a gradient elution. The analytes of interest were then eluted onto an analytical column for further chromatographic separation using a mobile phase of greater solvent strength. This method is specific and sensitive with a range of 3.75–1400 ng/ml for PMZ and 2.5–1400 ng/ml for the metabolites promethazine sulfoxide, monodesmethyl promethazine sulfoxide and monodesmethyl promethazine. The lower limits of quantitation (LLOQ) were 3.75 ng/ml with less than 6.2% C.V. for PMZ and 2.50 ng/ml with less than 11.5% C.V. for metabolites based on a signal-to-noise ratio of 10:1 or greater. The accuracy and precision were within ±11.8% in bias and not greater than 5.5% C.V. in intra- and inter-assay precision for PMZ and metabolites. Method robustness was investigated using a Plackett–Burman experimental design. The applicability of the analytical method for pharmacokinetic studies in humans is illustrated.     

Determination of betaine metabolites and dimethylsulfoniopropionate in coral tissues using liquid chromatography–time-of-flight mass spectrometry and stable isotope-labeled internal standards

A convenient procedure for determination of seven betaine analogs and dimethylsulfoniopropionate (DMSP) in extracts of coral tissues using LC–MS stable isotope dilution is described. Extraction procedures were optimized for selective extraction of polar metabolites from coral tissues. The LC–MS protocol employed a pentafluorophenylpropyl (PFPP) column for HPLC separation, with chromatographic resolution of isobaric and isomeric zwitterionic metabolites optimized by adjusting the acidity of the mobile phase. A ternary gradient was used to exploit the unusual retention characteristics of cationic metabolites on the PFPP column, with incorporation of ammonium acetate in a later gradient stage promoting elution of more hydrophobic betaines which are retained at high organic content in the absence of ammonium acetate. We demonstrate that the new LC–MS based method provides accurate measurements from nanomolar to high micromolar concentrations, and can be applied for profiling of betaine metabolites and DMSP in corals or other aquatic organisms.     

Rapid quantitation of free fatty acids in human plasma by high-performance liquid chromatography

We report a rapid and sensitive method for separation and quantitation of free fatty acids (FFAs) in human plasma using high-performance liquid chromatography (HPLC). Two established techniques of lipid extraction were investigated and modified to achieve maximal FFA recovery in a reasonably short time period. A modified Dole extraction method exhibited greater recovery (90%) and short processing times (30 min) compared to the method of Miles et al. Reversed-phase HPLC using UV detection was used for plasma FFA separation and quantitation. Two phenacyl ester derivatives, phenacyl bromide and p-bromophenacyl bromide, were investigated in order to achieve optimal separation of individual plasma FFAs (saturated and unsaturated) with desirable detection limits. Different chromatographic parameters including column temperature, column type and elution profiles (isocratic and gradient) were tested to achieve optimal separation and recovery of fatty acids. Phenacyl bromide esters of plasma fatty acids were best resolved using an octadecylsilyl column with endcapped silanol groups. An isocratic elution method using acetonitrile–water (83:17) at 2 ml/min with UV detection at 242 nm and a column temperature of 45°C was found to optimally resolve the six major free fatty acids present in human plasma (myristic [14:0], palmitic [16:0], palmitoleic [16:1], stearic [18:0], oleic [18:1] and linoleic [18:2]), with a run time of less than 35 min and detection limits in the nmol range. The entire process including plasma extraction, pre-column derivatization, and HPLC quantitation can be completed in 90 min with plasma samples as small as 50 μl. Over a wide physiological range, plasma FFA concentrations determined using our HPLC method agree closely with measurements using established TLC–GC methods (r²≥0.95). In addition, by measuring [¹⁴C] or [³H] radioactivity in eluent fractions following HPLC separation of plasma FFA, this method can also quantitate rates of FFA turnover in vivo in human metabolic studies employing isotopic tracers of one or more fatty acids.     

Separation and quantification of muropeptides with high-performance liquid chromatography

About 80 different muropeptides, the subunits which comprise the polymer murein of Escherichia coli, were resolved by high-performance liquid chromatography. The muropeptides were released from isolated murein by complete digestion with muramidase from Chalaropsis spec. The separation method is based on reversed phase chromatography of the sodium borohydride-reduced compounds using ODS (C18) columns and a linear gradient elution with sodium phosphate buffer and methanol as organic modifier. The effect of temperature, pH, ionic strength, and the steepness of the gradient and of different support materials on the separation of the muropeptides was investigated. The new method represents a major improvement over previous methods with respect to resolution, sensitivity, and speed. Analytical as well as preparative separations can be realized. Quantitative analysis of murein composition is achieved by a linear gradient from 50 mM sodium phosphate, pH 4.31, to 75 mM sodium phosphate, pH 4.95, containing 15% methanol for 135 min on a 250 X 4.6 mm 3-micron Hypersil ODS column at 55 degrees C using a flow rate of 0.5 ml/min. With uv detection at 205 nm about 20 micrograms of murein per analysis is sufficient. The detection limit per compound is about 5 ng. A method for the evaluation of the analytical data allowing a convenient comparison of different muropeptide pattern is described.     

Determination of a peptide-doxorubicin,prostate-specific antigen activated prodrug,and its active metabolites in human plasma using high-performance liquid chromatography with fluorescence detection. Stabilization of the peptide prodrug with EDTA

A method for the determination of I, a peptide-doxorubicin conjugate that was evaluated for the treatment of prostate cancer, and two of its active metabolites, doxorubicin and leucine-doxorubicin is described. Blood samples were chilled immediately after being drawn in order to prevent ex vivo entry of the metabolites into red blood cells. EDTA (10 mg/ml final concentration) was used to prevent plasma-mediated degradation of the peptide portion of the prodrug. After the addition of internal standard, plasma was prepared for analysis using a C-8 solid-phase extraction column. In order to overcome secondary ionic interactions with the silica-based extraction column, the analytes were eluted with ammonium hydroxide in methanol. The extracts were evaporated to dryness, reconstituted, and assayed by step change, gradient, reverse phase HPLC with fluorescence detection. Two interfering metabolites found in post dose plasma were chromatographically separated by an adjustment of the mobile phase pH. The within-day reproducibility of the doxorubicin and leucine-doxorubicin chromatographic retention times was improved by a brief washing of the analytical column with 90% acetonitrile after each injection. The range of the standard curve was 12.5-1250 ng/ml for doxorubicin and 25-2500 ng/ml for I and leucine-doxorubicin.     

Simultaneous determination of residual veterinary drugs in bovine, porcine, and chicken muscle using liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A simple and rapid method using liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/MS/MS) for the simultaneous determination of 130 veterinary drugs and their metabolites in bovine, porcine, and chicken muscle was developed. The drugs (1 to 10 ng/g, in muscle) were extracted from bovine, porcine, or chicken muscles with acetonitrile-methanol (95:5, v/v), and the extracts were delipidated with n-hexane saturated with acetonitrile. The extracts were evaporated, dissolved with methanol, analyzed by liquid chromatography with gradient elution on a C18 column, and determined by electrospray ionization tandem mass spectrometry. The detection limits ranged from 0.03 to 3 ng/g. The quantitation limits ranged from 0.1 to 10 ng/g. One hundred eleven, 122, and 123 drugs from bovine, porcine, and chicken muscle respectively showed recoveries between 70 and 110%.     

A rapid, high resolution high performance liquid chromatography profiling procedure for plant and microbial aromatic secondary metabolites

High performance liquid chromatography protocols have been developed to allow the simultaneous analysis of a very wide range of soluble aromatic secondary metabolites in unfractionated biological extracts. The methods are simple, sensitive, and highly reproducible. They are applicable to a wide variety of natural product investigations in both plants and microorganisms. High resolution of metabolites is achieved in 25 minutes by chromatography on a reverse phase C18 column in a gradient of 0 to 55% acetonitrile in water at pH 3. For example, near-baseline resolution of over 20 phenylpropanoid metabolites and 18 naturally occurring metabolites of indole-3-acetic acid can be obtained. The methods can be applied directly to whole tissue extracts without prepurification or enrichment. Moreover, the simplicity and sensitivity of the protocols allow their application to a large number of very small tissue samples, such as those encountered in research on host-microbe interactions. Such profiles allow one to monitor simultaneously the various alternative metabolic fates of a complex array of molecules. Examination of the profiles over time thus provides one with a powerful tool to correlate many concurrent molecular events that may relate to a given biological phenomenon. The final protocol requires as little as 1 milligram of tissue, which is extracted directly in a microfuge tube in 80% ethanol. With a variable wavelength detector, as little as 100 femtomoles of a given metabolite can be analyzed. Examples of the application of the protocols to a number of plant and microbial secondary product investigations and to screening for flavonoid mutants of Arabidopsis thaliana (L.) Heynh. are given.     

High performance liquid chromatographic-mass spectrometric determination of ginsenoside Rg3 and its metabolites in rat plasma using solid-phase extraction for pharmacokinetic studies

To support pharmacokinetic studies of ginsenosides, a novel method to quantitatively analyze ginsenoside Rg3 (Rg3), its prosapogenin ginsenoside Rh2 (Rh2) and aglycone 20(S)-protopanaxadiol (ppd) in rat plasma was developed and validated. The method was based on gradient separation of ginsenosides present in rat plasma using high performance liquid chromatography (HPLC), followed by detection with electrospray ionization(ESI) mass spectrometry (MS) in negative ion mode with the mobile phase additive, ammonium chloride (500 microM). Differentiation of ginsenosides was achieved through simultaneous detection of the [M(+)Cl(-)] adduct of ginsenoside Rg3 and [M(+)Cl(-)] adducts of its deglycosylated metabolites Rh2 and ppd, and other ions after solid phase extraction (SPE). The /specific ions monitored were m/z 819.50 for Rg3, m/z 657.35 for Rh2, m/z 495.40 for ppd and m/z 799.55 for the internal standard (digitoxin). The mean recoveries for Rg3, Rh2 and ppd were 77.85, 82.65 and 98.33%, respectively using 0.1 ml plasma for extraction. The lower limits of quantification were 10.0, 2.0 and 8.0 ng/ml (equivalent to 0.1, 0.02 and 0.08 ng in each 10 microl injection onto the HPLC column) for Rg3, Rh2 and ppd, respectively. The method has been demonstrated to be highly sensitive and accurate for the determination of Rg3 and its metabolites in rat plasma.     

Simultaneous determination of triclabendazole and its metabolites in bovine and goat tissues by liquid chromatography-tandem mass spectrometry

A sensitive liquid chromatography–tandem mass spectrometry method for the simultaneous determination of triclabendazole, its main metabolites (triclabendazole sulphone and triclabendazole sulphoxide) and a marker residue (ketotriclabendazole) in bovine and goat muscle, liver, and kidney samples is developed and validated. Analyte extraction from samples is effectively performed using liquid–liquid extraction by acetonitrile. Chromatographic separation is performed on a C₁₈ reversed-phase column with gradient elution. The analytes are detected by tandem quadrupole mass spectrometry after positive electrospray ionization by multiple reaction monitoring. The limits of detection for analytes are found to be 0.25–2.5 μg/kg in muscle tissues and 1–10 μg/kg in liver and kidney tissues, respectively. The recoveries of edible bovine and goat tissues range from 84.9% to 109.5% when spiked at different levels with analytes, with relative standard deviations generally below 12.8%.     

Quantitative detection of bisphenol A and bisphenol A diglycidyl ether metabolites in human plasma by liquid chromatography–electrospray mass spectrometry

Due to the ubiquity of epoxy resin compounds and their potential role in increasing the risk for reproductive dysfunction and cancer, the need for an assessment of human exposure is urgent. Therefore, we developed a method for measuring bisphenol A (BPA) and bisphenol A diglycidyl ether (BADGE) metabolites in human blood samples using high-performance liquid chromatography–electrospray ionization mass spectrometry (LC–MS). Human blood samples were processed using enzymatic deconjugation of the glucuronides followed by a novel sample preparation procedure using a solid-phase-cartridge column. This selective analytical method permits rapid detection of the metabolites, free BPA and a hydrolysis product of BADGE (BADGE-4OH) with detection limits in the low nanogram per milliliter range (0.1 ng ml⁻¹ of BPA and 0.5 ng ml⁻¹ of BADGE-4OH). The sample extraction was achieved by Oasis HLB column on gradient elution. The recoveries of BPA and BADGE-4OH added to human plasma samples were above 70.0% with a standard deviation of less than 5.0%. This selective, sensitive and accurate method will assist in elucidating potential associations between human exposure to epoxy-based compounds and adverse health effects.     

An automated SPE/LC/MS/MS method for the analysis of cocaine and metabolites in whole blood

As laboratories are called upon to develop novel, fast, and sensitive methods, here we present a completely automated method for the analysis of cocaine and its metabolites (benzoylecgonine, ecgonine methyl ester, ecgonine and cocaethylene) from whole blood. This method utilizes an online solid-phase extraction (SPE) with high performance liquid chromatographic separation and tandem mass spectrometric detection. Pretreatment of samples involve only protein precipitation and ultracentrifugation. An efficient online solid-phase extraction (SPE) procedure was developed using Hysphere MM anion sorbent. A gradient chromatography method with a Gemini C6-Phenyl (50mmx3.00mm i.d., 5microm) column was used for the complete separation of all components. Analysis was by positive ion mode electrospray ionization tandem mass spectrometry, using multiple reaction monitoring (MRM) to enhance the selectivity and sensitivity of the method. For the analysis, two MRM transitions are monitored for each analyte and one transition is monitored for each internal standard. With a 30-microL sample injection, linearity was analyte dependent but generally fell between 8 and 500ng/mL. The limits of detection (LODs) for the method ranged from 3 to 16ng/mL and the limits of quantitation (LOQs) ranged from 8 to 47ng/mL. The bias and precision were determined using a simple analysis of variance (ANOVA: single factor). The results demonstrate bias as <7%, and %precision as <9% for all components at each QC level.