Rapid determination of the active leflunomide metabolite A77 1726 in human plasma by high-performance liquid chromatography

A simple method for the measurement of the active leflunomide metabolite A77 1726 in human plasma by HPLC is presented. The sample workup was simple, using acetonitrile for protein precipitation. Chromatographic separation of A77 1726 and the internal standard, alpha-phenylcinnamic acid, was achieved using a C(18) column with UV detection at 305 nm. The assay displayed reproducible linearity for A77 1726 with determination coefficients (r2) > 0.997 over the concentration range 0.5-60.0 microg/ml. The reproducibility (%CV) for intra- and inter-day assays of spiked controls was <5%. The limit of quantification was 0.8 microg/ml. The average absolute recovery was approximately 100%. This assay is suitable for the determination of A77 1726 in plasma of patients taking leflunomide, and is simpler to use than other HPLC methods reported previously.     

Gas chromatographic determination of methamphetamine and its metabolite amphetamine in human plasma and urine following conversion to N-propyl derivatives

A gas chromatographic method for the simultaneous determination of methamphetamine and its metabolite amphetamine in human plasma and urine is described. The method utilizes reductive alkylation with propionaldehyde and sodium borohydride to produce N-propyl derivatives, which have excellent chromatographic properties. Structural analogs of the analytes, p-methylmethamphetamine and p-methylamphetamine, are used as internal standards. The method has good precision and accuracy for concentrations ranging from less than 10 ng/ml to 5000 ng/ml and has been used to measure plasma concentrations as part of a pharmacokinetic/pharmacodynamic study of methamphetamine in humans.     

Determination of the active metabolite of prulifloxacin in human plasma by liquid chromatography-tandem mass spectrometry

A liquid chromatographic-tandem mass spectrometric method (LC-MS/MS) for the determination of ulifloxacin, the active metabolite of prulifloxacin, in human plasma is described. After sample preparation by protein precipitation with methanol, ulifloxacin and ofloxacin (internal standard) were chromatographically separated on a C(18) column using a mobile phase consisting of methanol, water and formic acid (70:30:0.2, v/v/v) at a flow rate of 0.5 ml/min and then were detected using MS/MS by monitoring their precursor-to-product ion transitions, m/z 350-->m/z 248 for ulifloxacin and m/z 362-->m/z 261 for ofloxacin, in selected reaction monitoring (SRM) mode. Positive electrospray ionization was used for the ionization process. The linear range was 0.025-5.0 microg/ml for ulifloxacin with a lower limit of quantitation of 0.025 microg/ml. Within- and between-run precision was less than 6.6 and 7.8%, respectively, and accuracy was within 2.0%. The recovery ranged from 92.1 to 98.2% at the concentrations of 0.025, 0.50 and 5.0 microg/ml. Compared with the reported LC method, the present LC-MS/MS method can directly determine the ulifloxacin in human plasma without any need of derivatization. The present method has been successfully used for the pharmacokinetic studies of a prulifloxacin formulation product after oral administration to healthy volunteers.     

Rapid and selective high-performance liquid chromatographic method for the determination of metronidazole and its active metabolite in human plasma, saliva and gastric juice

A rapid and selective HPLC method has been developed for the separation and quantitation of metronidazole and its hydroxylated metabolite in human plasma, saliva and gastric juice. The assay requires a simple protein precipitation step prior to analysis and is selective, sensitive and reproducible. The limits of quantitation (0/5-ml sample) were at least 0.25 μg/ml for metronidazole and 0.20 μg/ml for its hydroxy metabolite. A Hypersil ODS 5 μm (150×4.6 mm I.D.) column was used with a mobile phase of acetonitrile-aqueous 0.05 M potassium phosphate buffer (pH 7) containing 0.1% triethylamine (10:90) delivered at a flow-rate of 1.0 ml/min.     

Liquid chromatography-mass spectrometry method for determination of ramipril and its active metabolite ramiprilat in human plasma

A fast and robust liquid chromatography-mass spectrometry (LC-MS-MS) method has been developed for simultaneous quantitation of the angiotensin-converting enzyme (ACE) inhibitor, ramipril and its metabolite ramiprilat in human plasma. The method involves a solid-phase extraction from plasma, simple isocratic chromatography conditions and mass spectrometric detection that enables a detection limit at sub-nanogram levels. The proposed method has been validated with a linear range of 0.5-250 ng/ml for both ramipril and ramiprilat. The overall recoveries for ramipril and ramiprilat were 88.7 and 101.8%, respectively.     

Stereoselective high-performance liquid chromatographic determination of ketamine and its active metabolite, norketamine, in human plasma

A stereoselective high-performance liquid chromatographic method for the determination of the enantiomers of ketamine and its active metabolite, norketamine, in human plasma is described. The compounds were extracted from plasma by liquid–liquid extraction three times in a combination of cyclohexane with 2.5 M NaOH, 1 mM HCl and 1 M carbonate buffer. Stereoselective separation was achieved on a Chiralcel OD column with a mobile phase of n-hexane–2-propanol (98:2, v/v). The detection wavelength was 215 nm. The lower limits of the determination of the method were 5 ng/ml for ketamine and 10 ng/ml for norketamine. The intra- and inter-day coefficients of variation ranged from 2.9 to 9.8% and from 3.4 to 10.7% for all compounds, respectively. The method was sensitive and sufficiently reproducible for stereoselective monitoring of ketamine and norketamine in human plasma during pharmacokinetic studies after the administration of ketamine for analgesia.     

Enantiomeric separation of tramadol and its active metabolite in human plasma by chiral high-performance liquid chromatography: application to pharmacokinetic studies

A sensitive and stereoselective high-performance liquid chromatographic assay for the quantitative determination of the analgesic tramadol and O-demethyltramadol, an active metabolite, is described in this work. Ketamine was used as internal standard. The assay involved a single tert-butymethylether extraction and liquid chromatography analysis with fluorescence detection. Chromatography was performed at 20 degrees C on a Chiracel OD-R column containing cellulose tris-(3,5-dimethylphenylcarbamate) as stationary phase, preceded by an achiral end-capped C18 column. The mobile phase was a mixture of phosphate buffer (containing sodium perchlorate (0.2 M) and triethylamine (0.09 M) adjusted to pH 6) and acetonitrile (80:20). The method developed was validated. The limit of quantitation of each enantiomer of tramadol and its active metabolite by this method was 0.5 ng/mL; only 0.5 mL of the plasma sample was required for the determination. The calibration curve was linear from 0.5 to 750 ng/mL for tramadol enantiomers, and from 0.5 to 500 ng/mL for O-demethyltramadol enantiomers. Intra and interday precision [coefficient of variation (CV)] did not exceed 10%. Mean recoveries of 95.95 and 97.87% for (+)R,R- and (-)S,S-tramadol and 97.70 and 98.79% for (+)R,R- and (-)S,S-O-demethyltramadol with CVs < 2.15% were obtained. Applicability of the method was demonstrated by a pharmacokinetic study in normal volunteers who received 100 mg of tramadol by the intravenous route.     

Immunoreactive and biologically active somatostatin in human and sheep milk

The presence of immunoreactive and biologically active somatostatin in sheep and human milk has been demonstrated. Milk somatostatin exhibits similar chromatographic behavior to that of synthetic somatostatin-14 on both reversed-phase C18 and cation-exchange high-performance liquid chromatography columns. Milk, in contrast to plasma, contains only somatostatin-14-like material. Milk somatostatin was capable of inhibiting the basal and the prostaglandin-induced release of growth hormone from anterior pituitary cell cultures in a pattern similar to synthetic somatostatin-14. The concentrations of the peptide, as determined by radioimmunoassay, were found to be 113 pg/ml in human milk and 150 +/- 4.8 pg/ml (mean +/- range) in sheep milk. These values are severalfold higher than the corresponding concentration of the peptide in the plasma of these species. These findings are analogous to our previous observations concerning two other hypothalamic hormones, luliberin and thyroliberin [Baram, T., Koch, Y., Hazum, E. and Fridkin, M. (1977) Science (Wash. DC) 198, 300-302]. The high concentration of somatostatin and other neuropeptides in milk implies either an active concentrating mechanism in the mammary gland or an additional extrahypothalamic source for the synthesis and release of these peptides.     

Rapid determination of PEGylated liposomal doxorubicin and its major metabolite in human plasma by ultraviolet-visible high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the quantification of doxorubicin derived from PEGylated liposomal doxorubicin (Doxil) and its major metabolite in human plasma. This method utilizes Triton X-100 to disperse the liposome, followed by a protein precipitation step with 5-sulfosalicylic acid. Analytes in the resultant supernatant are separated on a Discovery RP amide C(16) column (250 x 3 mm I.D., 5 microm) using an isocratic elution with a mobile phase consisting of 0.05 M sodium acetate (pH 4.0) and acetonitrile (72:28). The retention times for doxorubicin and the internal standard daunorubicin were 4.8 and 10.1 min, respectively. The column eluate was monitored by UV-visible detection at 487 nm. The determination of doxorubicin was found to be linear in the range of 1.0 ng/mL to 25 microg/mL, with intra-day and inter-day coefficients of variation and percent error < or =10%. The recovery of doxorubicin from plasma was >69.3%, with a liposomal dispersion efficiency of >95.7%. Our analytical method for free and PEGylated doxorubicin in human plasma is rapid, avoids organic extractions, and maintains sensitivity for the parent compound and its major metabolite, doxorubicinol.     

Demonstration of enzymatic conversion of lysolecithin to lecithin in normal human plasma

An enzyme in normal human plasma that converts [1-acyl ¹⁴C] lysolecithin to lecithin is demonstrated. This enzyme is inhibited by heparin and is not derived from platelets or other blood elements. The synthesis of lecithin from labeled lysolecithin was not stimulated by ATP and CoA or by oleyl CoA and there was nearly an equal distribution of labeled fatty acid between the two positions of lecithin indicating that the enzyme may be a lysolecithin: lysolecithin acyl transferase (LLAT). The enzyme is associated with the lipoproteins of the plasma, and may have a physiological role in the formation of saturated cholesterol esters in plasma.     

Concanavalin a promotes the uptake of lysosomal hydrolases by human fibroblasts

Human placental hexosaminidase B and β-galactosidase are taken up very poorly by human fibroblasts in culture. However, if fibroblasts manifesting genetically determined deficiencies of these lysosomal hydrolases are first treated with concanavalin A, then enzyme uptake is markedly increased. Enzyme activity which becomes associated with concanavalin A-treated fibroblasts maintained at 4°C can be greatly removed by treatment with haptene sugar, while enzyme activity which becomes associated with cells maintained at 37°C is refractory to haptene treatment. These results are interpreted as an initial binding of enzyme to concanavalin A molecules located at the cell surface, followed by an active cellular process leading to internalization of the lectin-enzyme complexes.     

Analysis of ifosforamide mustard, the active metabolite of ifosfamide, in plasma

Ifosforamide mustard is the active metabolite of ifosfamide, a cytostatic drug. In this study a sensitive and selective method for the analysis of ifosforamide mustard in plasma is described. The method consists of direct derivatisation of ifosforamide mustard in plasma with diethyldithiocarbamate and subsequent solid-phase extraction of the resulting derivative. The analysis of the derivatisation product was performed by high-performance liquid chromatography with UV detection. The calibration graph was linear in the concentration range 0.45–45 μM and the minimum detectable concentration was 0.45 μmol. The samples were stabilised by addition of semicarbazide and sodium chloride. A patient's plasma sample was analysed by means of the described method. The ifosforamide mustard concentration was 2.3 μM.     

Rapid determination of gefitinib and its main metabolite, O-desmethyl gefitinib in human plasma using liquid chromatography-tandem mass spectrometry

A novel, rapid and specific liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the simultaneous quantification of gefitinib and its predominant metabolite, O-desmethyl gefitinib in human plasma. Chromatographic separation of analytes was achieved on an Alltima C18 analytical HPLC column (150 mm × 2.1 mm, 5 μm) using an isocratic elution mode with a mobile phase comprised acetonitrile and 0.1% formic acid in water (30:70, v/v). The flow rate was 300 μL/min. The chromatographic run time was 3 min. The column effluents were detected by API 4000 triple quadrupole mass spectrometer using electrospray ionization (ESI) in positive mode. Linearity was demonstrated in the range of 5-1000 ng/mL for gefitinib and 5-500 ng/mL for O-desmethyl gefitinib. The intra- and inter-day precisions for gefitinib and O-desmethyl gefitinib were ≤10.8% and the accuracies ranged from 89.7 to 104.7% for gefitinib and 100.4 to 106.0% for O-desmethyl gefitinib. This method was used as a bioanalytical tool in a phase I clinical trial to investigate the possible effect of hydroxychloroquine on the pharmacokinetics of gefitinib. The results of this study enabled clinicians to ascertain the safety of the combination therapy of hydroxychloroquine and gefitinib in patients with advanced (Stage IIIB-IV) non-small cell lung cancer (NSCLC).     

Simultaneous determination of quinapril and its active metabolite quinaprilat in human plasma using high-performance liquid chromatography with ultraviolet detection

A high-performance liquid chromatography (HPLC) procedure for the simultaneous determination of quinapril and its active metabolite quinaprilat in human plasma samples is described. A one-step solid-phase extraction (SPE) with C18 cartridges was coupled with a reversed-phase HPLC system. The system requires two mobile phases composed of tetrabutyl ammonium hydrogensulfate (10 mM adjusted to pH 7)-acetonitrile (62:38, v/v) for quinapril, and (25:75, v/v) for quinaprilat elution through a C18 Symmetry column and detection at a wavelength of 215 nm. Calibration curves were linear over the ranges 20 to 1,000 ng/ml for quinaprilat and 10 to 500 for quinapril. The limits of quantification were 20 and 10 ng/ml for quinaprilat and quinapril, respectively. Extraction recoveries were higher than 90% for quinapril and 80% for quinaprilat. This method has been successfully applied to a bioequivalence study of quinapril in healthy subjects.     

Differences between human plasma and serum metabolite profiles

Background

Human plasma and serum are widely used matrices in clinical and biological studies. However, different collecting procedures and the coagulation cascade influence concentrations of both proteins and metabolites in these matrices. The effects on metabolite concentration profiles have not been fully characterized.

Methodology/Principal Findings

We analyzed the concentrations of 163 metabolites in plasma and serum samples collected simultaneously from 377 fasting individuals. To ensure data quality, 41 metabolites with low measurement stability were excluded from further analysis. In addition, plasma and corresponding serum samples from 83 individuals were re-measured in the same plates and mean correlation coefficients (r) of all metabolites between the duplicates were 0.83 and 0.80 in plasma and serum, respectively, indicating significantly better stability of plasma compared to serum (p = 0.01). Metabolite profiles from plasma and serum were clearly distinct with 104 metabolites showing significantly higher concentrations in serum. In particular, 9 metabolites showed relative concentration differences larger than 20%. Despite differences in absolute concentration between the two matrices, for most metabolites the overall correlation was high (mean r = 0.81±0.10), which reflects a proportional change in concentration. Furthermore, when two groups of individuals with different phenotypes were compared with each other using both matrices, more metabolites with significantly different concentrations could be identified in serum than in plasma. For example, when 51 type 2 diabetes (T2D) patients were compared with 326 non-T2D individuals, 15 more significantly different metabolites were found in serum, in addition to the 25 common to both matrices.

Conclusions/Significance

Our study shows that reproducibility was good in both plasma and serum, and better in plasma. Furthermore, as long as the same blood preparation procedure is used, either matrix should generate similar results in clinical and biological studies. The higher metabolite concentrations in serum, however, make it possible to provide more sensitive results in biomarker detection.     

Simultaneous determination of dronedarone and its active metabolite debutyldronedarone in human plasma by liquid chromatography-tandem mass spectrometry: application to a pharmacokinetic study

Dronedarone is a derivative of amiodarone--a popular antiarrhythmic drug. It was developed to overcome the limiting iodine-associated toxicities of amiodarone. Debutyldronedarone is a major circulating active metabolite of dronedarone in humans. To investigate the pharmacokinetics of dronedarone, a rapid, simple, and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneously determine dronedarone and debutyldronedarone in human plasma using amiodarone as internal standard (IS). Acetonitrile with IS was used to precipitate proteins from a 50-μL aliquot of plasma. Effective chromatographic separation was performed on a CAPCELL PAK C(18) MG (100 mm × 4.6 mm, 5 μm) column with gradient elution (5 mmol/L ammonium acetate-acetonitrile, with each phase containing 0.2% acetic acid) at a flow rate of 0.7 mL/min. Complete separation was achieved within 5.5 min. Detection was carried out on an tandem mass spectrometer in multiple reaction monitoring mode using a positive atmospheric pressure chemical ionization interface. A lower limit of quantification of 0.200 ng/mL was achieved for both dronedarone and debutyldronedarone, with acceptable precision and accuracy. The linear range of the method was from 0.200 to 200 ng/mL for each analyte. Intra- and inter-day precisions were lower than 7.2% in relation to relative standard deviation, while accuracy was within ±5.1% in terms of relative error for analytes. Our findings demonstrate the successful application of the validated LC-MS/MS method to a pharmacokinetic study after a single oral administration of 400mg dronedarone to six healthy volunteers.     

Mutants of Escherichia coli K12 unable to grow on non-fermentable carbon substrates

Among a number of mutants unable to utilize non-fermentable carbon substrates, scoring for membrane ATPase and for ATP-driven transhydrogenase activity permitted to distinguish two phenotypes: (A) mutants lacking ATPase and ATPdriven transhydrogenase; (B) one mutant with an ATPase which behaved according to several criteria as released into solution instead of being membrane bound, a.o it exhibited no ATP-driven transhydrogenase activity. All A and B mutants exhibited a common nutritional pattern.The ATPase-deficient group, when scored for ATPase-binding sites on its membrane particles revealed three different subgroups: (1) mutants having free ATPase-binding sites, (2) mutants with ATPase-binding sites made available by the procedure which releases ATPase from wild-type membrane, and (3) mutants with no detectable ATPase-binding sites.Membranes of the mutant B with unbound ATPase also exhibited a deficiency in ATPase-binding sites, but its soluble ATPase was also found unable to bind to ATPase-binding sites of wild type membranes.The double alteration, namely abnormal or inactive ATPase and absence of ATPase-binding sites on the membrane is compatible with a single mutational defect.