Achiral and chiral high-performance liquid chromatographic methods for clinafloxacin, a fluoroquinolone antibacterial, in human plasma

Achiral and chiral HPLC methods were developed for clinafloxacin, a quinolone antimicrobial agent. For achiral assay, analytes were isolated from plasma by precipitating plasma proteins. Separation was achieved on a C₁₈ column using an isocratic eluent of ion pairing solution–acetonitrile (80:20, v/v) at 1.0 ml/min with UV detection at 340 nm. The ion pairing solution was 0.05 M citric acid, 1.15 mM tetrabutylammonium hydroxide and 0.1% ammonium perchlorate. Inter-assay accuracy was within 4.9% with an inter-assay precision of 3.7% over a quantitation range of 0.025 to 10.0 μg/ml. For chiral assay, analytes were isolated from plasma by solid-phase extraction. Separation was achieved on a Crownpak CR(+) column using an isocratic eluent of water–methanol (88:12, v/v) containing 0.1 mM decylamine at 1.0 ml/min with UV detection at 340 nm. Perchloric acid was added to adjust pH to 2. Inter-assay accuracy was within 3.5% with a inter-assay precision of 5.4% over a quantitation range of 0.040 to 2.5 μg/ml.     

A validated high-performance liquid chromatographic assay for the simultaneous determination of denaverine and its N-monodemethyl metabolite in human plasma

An isocratic reversed-phase high-performance liquid chromatographic method for the simultaneous determination of denaverine and its N-monodemethyl metabolite (MD 6) in human plasma is described. The assay involves the extraction with an n-heptane–2-propanol mixture (9:1, v/v) followed by back extraction into 12.5% (w/w) phosphoric acid. The analytes of interest and the internal standard were separated on a Superspher RP8 column using a mobile phase of acetonitrile–0.12 M NH₄H₂PO₄–tetrahydrofuran (24:17.2:1, v/v), adjusted to pH 3 with 85% (w/w) phosphoric acid. Ultraviolet detection was used at an operational wavelength of 220 nm. The retention times of MD 6, denaverine and the internal standard were 5.1, 6.3 and 10.2 min, respectively. The assay was validated according to international requirements and was found to be specific, accurate and precise with a linear range of 2.5–150 ng/ml for denaverine and MD 6. Extraction recoveries for denaverine and MD 6 ranged from 44 to 49% and from 42 to 47%, respectively. The stability of denaverine and MD 6 in plasma was demonstrated after 24 h storage at room temperature, after three freeze–thaw cycles and after 7 months frozen storage below −20°C. The stability of processed samples in the autosampler at room temperature was confirmed after 24 h storage. The analytical method has been applied to analyses of plasma samples from a pharmacokinetic study in man.     

On the specificity of the alcoholic,acidic silver nitrate reagent for the histochemical localization of ascorbic acid

Summary The defects besetting the histochemical localization of ascorbic acid were removed in the modified method described here by the simultaneous fixation of the experimental material and its reaction with silver nitrate by the use of alcoholic, acidic silver nitrate reagent in the dark at 0–3°C for 24 hours or longer at pH 2–2.5.The fixatives like acetic acid and alcohol of the reagent ensure quick penetration of AgNO₃ for fixation of ascorbic acid in situ before sectioning. It has been experimentally established that none of the other reductants react with AgNO₃ at the pH and the temperature mentioned.The sections were devitaminized by treatment with 6–10% formaline for 3–4 hours to serve as a control.     

Liquid chromatographic–mass spectrometric determination of celecoxib in plasma using single-ion monitoring and its use in clinical pharmacokinetics

Celecoxib is a cyclooxygenase-2 specific inhibitor, that has been recently and intensively prescribed as an anti-inflammatory drug in rheumatic osteoarthiritis. A robust, highly reliable and reproducible liquid chromatographic–mass spectrometric assay is developed for the determination of celecoxib in human plasma using sulindac as an internal standard. The run cycle-time is <4 min. The assay method involved extraction of the analytes from plasma samples at pH 5 with ethyl acetate and evaporation of the organic layer. The reconstituted solution of the residue was injected onto a Shim Pack GLC-CN, C₁₈ column and chromatographed with a mobile phase comprised of acetonitrile–1% acetic acid solution (4:1) at a flow-rate of 1 ml/min. The mass spectrometer (LCQ Finnigan Mat) was programmed in the positive single-ion monitoring mode to permit the detection and quantitation of the molecular ions of celecoxib and sulindac at m/z 382 and 357, respectively. The peak area ratio of celecoxib/sulindac and concentration are linear (r²>0.994) over the concentration range 50–1000 ng/ml with a lowest detection limit of 20 ng/ml of celecoxib. Within- and between-day precision are within 1.58–4.0% relative standard deviation and the accuracy is 99.4–107.3% deviation of the nominal concentrations. The relative recoveries of celecoxib from human plasma ranged from 102.4 to 103.3% indicating the suitability of the method for the extraction of celecoxib and I.S. from plasma samples. The validated LC–MS method has been utilized to establish various pharmacokinetic parameters of celecoxib following a single oral dose administration of celecoxib capsules in two selected volunteers.     

Water conservation and protein metabolism in northern elephant seal pups during the postweaning fast

Urine production and N output were monitored in northern elephant seal (Mirounga angustirostris) pups progressing through 10 weeks of a natural postweaning fast. Urine output declind by 84% (to 69±12 ml·day^–1) at 10 weeks (P<0.05). Glomerular filtration rate at 10 weeks was 51% of the 67±3 ml serum·min^–1 observed during week 1 (P<0.05). Urine N excretion fell by 69% to 1.2±0.17 g·day^–1, while urinary concentration increased (P<0.05). Serum urea declined from an initial 11 mmol·1^–1 to 5–7 mmol·1^–1 by 5 weeks. The fall in urinary N loss (and thus amino acid oxidation) was concomitant with depressed metabolic rate. Therefore, protein contributed little toward meeting energy demands (i.e., <4% of average metabolic rate) throughout fasting. These data indicate that fasting pups improve water conservation and minimize protein catabolism during prolonged natural fasts without an exogenous source of water.Abbreviations AA amino acid(s) - AMR average metabolic rate - ANOVA one-way analysis of variance - BMR basal metabolic rate - BUN blood urea nitrogen - EP end product - EWL evaporative water loss - [Gr]_s serum creatinine concentration - GFR glomerular filtration rate - LBM lean body mass - LML Long Marine Laboratory - MR metabolic rate - NEFA non-esterified fatty acids - RMR resting metabolic rate - TCA tricarboxylic acid - U:C ulinary urea: creatinine concentration ratio     

High-performance liquid chromatographic determination of trimebutine and its major metabolite, N-monodesmethyl trimebutine, in rat and human plasma

A rapid, selective and very sensitive ion-pairing reversed-phase HPLC method was developed for the simultaneous determination of trimebutine (TMB) and its major metabolite, N-monodesmethyltrimebutine (NDTMB), in rat and human plasma. Heptanesulfonate was employed as the ion-pairing agent and verapamil was used as the internal standard. The method involved the extraction with a n-hexane–isopropylalcohol (IPA) mixture (99:1, v/v) followed by back-extraction into 0.1 M hydrochloric acid and evaporation to dryness. HPLC analysis was carried out using a 4-μm particle size, C₁₈-bonded silica column and water–sodium acetate–heptanesulfonate–acetonitrile as the mobile phase and UV detection at 267 nm. The chromatograms showed good resolution and sensitivity and no interference of plasma. The mean recoveries for human plasma were 95.4±3.1% for TMB and 89.4±4.1% for NDTMB. The detection limits of TMB and its metabolite, NDTMB, in human plasma were 1 and 5 ng/ml, respectively. The calibration curves were linear over the concentration range 10–5000 ng/ml for TMB and 25–25000 ng/ml for NDTMB with correlation coefficients greater than 0.999 and with within-day or between-day coefficients of variation not exceeding 9.4%. This assay procedure was applied to the study of metabolite pharmacokinetics of TMB in rat and the human.     

Sensitive, high-throughput gas chromatographic–mass spectrometric assay for fluoxetine and norfluoxetine in human plasma and its application to pharmacokinetic studies

A sensitive, robust gas chromatographic–mass spectrometric assay suitable for use in pharmacokinetic or bioequivalence studies is presented for the selective serotonin reuptake inhibitor, fluoxetine, and its major metabolite, norfluoxetine (N-desmethylfluoxetine). This method employs solid-phase extraction followed by acetylation with trifluoroacetic anhydride and analysis of the derivatives using selected ion monitoring. The lower limit of quantification was 1.0 ng/ml, and the assay was linear for both analytes from 1 to 100 ng/ml. Mean recoveries following solid-phase extraction at concentrations of 5.0, 20 and 100 ng/ml were 91% (fluoxetine) and 87% (norfluoxetine). Assay precision (as mean RSD) and accuracy (as mean relative error) for both analytes were tested at the same three nominal concentrations and were found to be within 10% in all cases. Analysis of fluoxetine concentrations in plasma samples from 18 volunteers following administration of a single 40 mg dose of fluoxetine provided the following pharmacokinetic data (mean±SD): C_max, 32.73±9.21 ng/ml; AUC_0–∞, 1627±1372 ng/ml h; T_max, 3.08 h (median); k_e, 0.022±0.007 h⁻¹; elimination half-life, 37.69±21.70 h.     

Method for the simultaneous determination of losartan and its major metabolite, EXP-3174, in human plasma by liquid chromatography–electrospray ionization tandem mass spectrometry

A liquid chromatography–electrospray ionization tandem mass spectrometric method was developed for the simultaneous determination of losartan and its major active metabolite, EXP-3174, in human plasma. The two analytes and the internal standard (DuP-167) were extracted from plasma under acidic conditions by using solid-phase extraction cartridges containing a sorbent of copolymer, poly(divinylbenzene-co-N-vinylpyrrolidone). The analytes were separated by LC equipped with a reversed-phase C₁₈ column, and introduced into the mass spectrometer via the electrospray ion source with pneumatically-assisted nebulization. For LC–MS–MS samples, an isocratic mobile phase consisting of [0.1% triethylamine–0.1% acetic acid (pH 7.1)]–acetonitorile (65:35, v/v) was used, and the assay was monitored for the negative fragment ions of the analytes. The method demonstrated linearity from 1 to 1000 ng/ml for both losartan and EXP-3174. The limit of quantification for both compounds in plasma was 1 ng/ml. This assay method may be useful for the measurement of levels of the two compounds in clinical studies of losartan.     

Determination of clindamycin in human plasma by liquid chromatography–electrospray tandem mass spectrometry: application to the bioequivalence study of clindamycin

A simple, sensitive and specific liquid chromatography–electrospray tandem mass spectrometry (LC–MS–MS) method for the determination of clindamycin (I) was developed. Both I and verapamil (II, internal standard) were analyzed using a C₁₈ column with a mobile phase of 80% acetonitrile–0.01% trifluoroacetic acid. Column eluents were monitored by electrospray tandem mass spectrometry. Multiple reaction monitoring (MRM) using the parent to daughter combinations of m/z 425→126 and 455→165 was used to quantitate I. A limit of quantitation of 0.0500 μg/ml was found. The assay exhibited a linear dynamic range of 0.0500–20.0 μg/ml and gave a correlation coefficient (r²) of 0.998 or better. The chromatographic run time was approximately 2 min. The intra-batch precision and accuracy of the quality controls (QCs, 0.0500, 0.150, 1.50, 15.0 and 20.0 μg/ml) were characterized by coefficients of variation (CVs) of 5.13 to 13.7% and relative errors (REs) of −4.34 to 4.58%, respectively. The inter-batch precision and accuracy of the QCs were characterized by CVs of 4.35 to 8.32% and REs of −10.8 to −4.17%, respectively. The method has successfully been applied to the analysis of samples taken up to 12 h after oral administration of 300 mg of I in healthy volunteers.     

Stereospecific high-performance liquid chromatographic assay of metoprolol

A valid, sensitive high-performance liquid chromatographic technique is reported for the separation of the two enantiomers of metoprolol in human plasma. The procedure involves pre-column derivatization with the homochiral reagent S-(+)-1-(1-naphthyl)-ethyl isocyanate. Once formed, the diastereomers are separated using normal-phase high-performance liquid chromatography. Fluorescence detection (220 nm excitation; no emission filter) was utilized, resulting in baseline resolution (R_s > 1.5). The peaks corresponding to metoprolol enantiomers were free from interference throughout the examined range of 5–500 ng/ml; accuracy and precision were within approximately 10%. Analysis of a plasma sample collected from a healthy volunteer demonstrated that the assay is applicable to clinical studies.     

Resolution and quantitation of pentazocine enantiomers in human serum by reversed-phase high-performance liquid chromatography using sulfated β-cyclodextrin as chiral mobile phase additive and solid-phase extraction

A sensitive and stereospecific HPLC method was developed for the analysis of (−)- and (+)-pentazocine in human serum. The assay involves the use of a phenyl solid-phase extraction column for serum sample clean-up prior to HPLC analysis. Chromatographic resolution of the pentazocine enantiomers was performed on a octadecylsilane column with sulfated-β-cyclodextrin (S-β-CD) as the chiral mobile phase additive. The composition of the mobile phase was aqueous 10 mM potassium dihydrogenphosphate buffer pH 5.8 (adjusted with phosphoric acid)–absolute ethanol (80:20, v/v) containing 10 mM S-β-CD at a flow-rate of 0.7 ml/min. Recoveries of (−)- and (+)-pentazocine were in the range of 91–93%. Linear calibration curves were obtained in the 20–400 ng/ml range for each enantiomer in serum. The detection limit based on S/N=3 was 15 ng/ml for each pentazocine enantiomer in serum with UV detection at 220 nm. The limit of quantitation for each enantiomer was 20 ng/ml. Precision calculated as R.S.D. and accuracy calculated as error were in the range 0.9–7.0% and 1.2–6.2%, respectively, for the (−)-enantiomer and 0.8– 7.6% and 1.2–4.6%, respectively, for the (+)-enantiomer (n=3).     

Analysis of N-methylhistamine by gas chromatography–mass spectrometry

A gas chromatography–electron capture mass spectrometry assay has been developed for the histamine H₃ receptor agonist, N^α-methylhistamine (N^α-MH). The assay is linear from 50 pg–10 ng, with a limit of detection of 50 pg/ml for gastric juice and plasma, and 50 pg/sample for bacteria (10⁷–10⁸ CFU) and gastric tissue (5–10 mg wet weight). The limits of quantification are 100 pg/ml for gastric juice (%RSD=1.4) and plasma (%RSD=9.4), and 100 pg/sample for bacteria (%RSD=3.9) and tissue (%RSD=5.8). N^α-MH was not present in human plasma, but low levels (1.4 ng/ml and 0.4 ng/ml) were detected in two samples of human gastric juice obtained from patients infected with Helicobacter pylori.     

Determination of bezafibrate, ciprofibrate and fenofibric acid in human plasma by high-performance liquid chromatography

A selective high-performance liquid chromatographic method to assess either bezafibrate, ciprofibrate or fenofibric acid plasma levels is described. Drugs are extracted with diethyl ether, after acidification with HCl. An isocratic acetonitrile-0.02 M H₃PO₄ (55:45) mobile phase, a C₁₈ (5 μm) column and UV detection are used. The LOQ found was 0.25 μg/ml for the three fibrates. Intra- and inter-assay accuracy ranges were 90–107% and 82–111%; 96–115% and 94–107%; 94–114% and 94–126% for bezafibrate, ciprofibrate and fenofibric acid, respectively. Intra- and inter-assay precision (C.V.% ranges) were 1.72–3.06% and 2.66–7.67%; 1.88–4.64% and 0.62–2.99%; 1.26–4.69% and 3.56–7.17% for the three fibrates studied. Its sensitivity, accuracy and precision make it a useful tool for monitoring plasma levels of these drugs in a clinical setting and for research purposes.     

Determination of ceftazidime in plasma using high-performance liquid chromatography and electrochemical detection: Application for individualizing dosage regimens in elderly patients

This study describes a sensitive HPLC–electrochemical detection method for the analysis of ceftazidime, a third-generation cephalosporin, in human plasma. The extraction procedure involved protein precipitation with 30% trichloroacetic acid. The separation was achieved on a reversed-phase column (250×4.6 mm I.D., 5 μm) packed with C₁₈ Kromasil with isocratic elution and a mobile phase consisting of acetonitrile–25 mM KH₂PO₄–Na₂HPO₄ buffer, pH 7.4 (10:90, v/v). The proposed analytical method is selective, reproducible and reliable. The assay has a precision of 0.2–15.1% (C.V.) in the range of 5–200 μg ml⁻¹. (corresponding to 0.5 to 20 ng of ceftazidime injected onto the column), and is optimised for assaying 50 μl of plasma. The extraction recovery from plasma was approximately 100%. The method was highly specific for ceftazidime and there was no interference from either commonly administered drugs or endogenous compounds. This assay was used to measure ceftazidime in elderly patients for therapeutic drug monitoring.     

Improved coupled column liquid chromatographic method for high-speed direct analysis of urinary trans,trans-muconic acid, as a biomarker of exposure to benzene

A coupled column liquid chromatographic (LC–LC) method for high-speed analysis of the urinary ring-opened benzene metabolite, trans,trans-muconic acid (t,t-MA) is described. Efficient on-line clean-up and concentration of t,t-MA from urine samples was obtained using a 3 μm C₁₈ column (50×4.6 mm I.D.) as the first column (C-1) and a 5 μm C₁₈ semi-permeable surface (SPS) column (150×4.6 mm I.D.) as the second column (C-2). The mobile phases applied consisted, respectively, of methanol–0.05% trifluoroacetic acid (TFA) in water (7:93, v/v) on C-1, and of methanol–0.05% TFA in water (8:92, v/v) on C-2. A rinsing mobile phase of methanol–0.05% TFA in water (25:75, v/v) was used for cleaning C-1 in between analysis. Under these conditions t,t-MA eluted 11 min after injection. Using relatively non-specific UV detection at 264 nm, the selectivity of the assay was enhanced remarkably by the use of LC–LC allowing detection of t,t-MA at urinary levels as low as 50 ng/ml (S/N>9). The study indicated that t,t-MA analysis can be performed by this procedure in less than 20 min requiring only pH adjustment and filtration of the sample as pretreatment. Calibration plots of standard additions of t,t-MA to blank urine over a wide concentration range (50–4000 ng/ml) showed excellent linearity (r>0.999). The method was validated using urine samples collected from rats exposed to low concentrations of benzene vapors (0.1 ppm for 6 h) and by repeating most of the analyses of real samples in the course of measurement sequences. Both the repeatability (n=6, levels 64 and 266 ng/ml) and intra-laboratory reproducibility (n=6, levels 679 and 1486 ng/ml) were below 5%.     

Rapid determination of ranitidine in human plasma by high-performance liquid chromatography without solvent extraction

A simple high-performance liquid chromatographic procedure was developed for the determination of ranitidine in human plasma. The method entailed direct injection of the plasma samples after deproteination using perchloric acid. The chromatographic separation was accomplished with an isocratic elution using mobile phase consisting of 21 mM disodium hydrogen phosphate–triethylamine-acetonitrile (1000:60:150, v/v), pH 3.5. Analyses were run at a flow-rate of 1.3 ml/min using a μbondapak C₁₈ column and ultraviolet detection at a wavelength of 320 nm. The method was specific and sensitive, with a quantification limit of approximately 20 ng/ml and a detection limit of 5 ng/ml at a signal-to-noise ratio of 3:1. The mean absolute recovery was about 96%, while the within- and between-day coefficient of variation and percent error values of the assay method were all less than 8%. The linearity was assessed in the range of 20–1000 ng/ml plasma, with a correlation coefficient of greater than 0.999. This method has been used to analyze several hundred human plasma samples for bioavailability studies.     

o-Phthalaldehyde–N-acetylcysteine polyamine derivatives: formation and stability in solution and in C18 supports

A comparative study of different derivatization procedures has been performed in order to improve the stability of the reaction products o-phthalaldehyde–N-acetylcysteine (OPA–NAC) polyamines. Procedures such as solution derivatization, solution derivatization followed by retention on a packing support, derivatization on different packing supports and on-column derivatization, have been optimized and compared. The degradation rate constant (k) of the derivative was dependent on the procedure used and on the analyte. For the spermine (the most unstable isoindol tested) k was 8±2×10⁻² min⁻¹ in solution versus 7.7±1.1×10⁻⁴ min⁻¹ on the (C₁₈) solid support. The results obtained showed that forming the derivative on the packing support (C₁₈) gave the best results following this procedure: conditioning the cartridges with borate buffer (1 ml, 0.5 M, pH 8), retention of the analyte, addition of 0.8 ml of OPA–NAC reagent, 0.2 ml borate buffer 0.8 M (pH 8) and elution of the isoindol with 3 ml of MeOH–borate buffer (9:1). The different derivatization procedures have been used to study the stability of the reaction products OPA–NAC polyamines formed in urine matrix using spermine as model compound. Similar results were obtained for standard solutions and urine samples.     

Production of gluconic acid by immobilized in pumice stones mycelium of Aspergillus niger using unconventional oxygenation of culture

Gluconic acid was produced in repeated batch processes with Aspergillus niger AM-11, immobilized in pumice stone particles using an unconventional oxygenation of culture media based on the addition of H₂O₂, decomposed by catalase to O₂ and water. The highest gluconic acid productivity of 8.2 g l^–1 h^–1 was reached with 30 g immobilized mycelium per 150 ml, 10% (w/v) glucose, at 24 °C and pH 6.5, with O₂ at 100% saturation. The immobilized mycelium was successfully reused up to 8 times in 1-h batches with only a slight loss (11%) of gluconic acid productivity.     

Determination of celecoxib in human plasma and rat microdialysis samples by liquid chromatography tandem mass spectrometry

Methods for the determination of celecoxib in human plasma and rat microdialysis samples using liquid chromatography tandem mass spectrometry are described. Celecoxib and an internal standard were extracted from plasma by solid-phase extraction with C₁₈ cartridges. Thereafter compounds were separated on a short narrow bore RP C₁₈ column (30×2 mm). Microdialysis samples did not require extraction and were injected directly using a narrow bore RP C₁₈ column (70×2 mm). The detection was by a PE Sciex API 3000 mass spectrometer equipped with a turbo ion spray interface. The compounds were detected in the negative ion mode using the mass transitions m/z 380→316 and m/z 366→302 for celecoxib and internal standard, respectively. The assay was validated for human plasma over a concentration range of 0.25–250 ng/ml using 0.2 ml of sample. The assay for microdialysis samples (50 μl) was validated over a concentration range of 0.5–20 ng/ml. The method was utilised to determine pharmacokinetics of celecoxib in human plasma and in rat spinal cord perfusate.