New validated method for piracetam HPLC determination in human plasma

The new method for HPLC determination of piracetam in human plasma was developed and validated by a new approach. The simple determination by UV detection was performed on supernatant, obtained from plasma, after proteins precipitation with perchloric acid. The chromatographic separation of piracetam under a gradient elution was achieved at room temperature with a RP-18 LiChroSpher 100 column and aqueous mobile phase containing acetonitrile and methanol. The quantitative determination of piracetam was performed at 200 nm with a lower limit of quantification LLQ=2 microg/ml. For this limit, the calculated values of the coefficient of variation and difference between mean and the nominal concentration are CV%=9.7 and bias%=0.9 for the intra-day assay, and CV%=19.1 and bias%=-7.45 for the between-days assay. For precision, the range was CV%=1.8/11.6 in the intra-day and between-days assay, and for accuracy, the range was bias%=2.3/14.9 in the intra-day and between-days assay. In addition, the stability of piracetam in different conditions was verified. Piracetam proved to be stable in plasma during 4 weeks at -20 degrees C and for 36 h at 20 degrees C in the supernatant after protein precipitation. The new proposed method was used for a bioequivalence study of two medicines containing 800 mg piracetam.     

Determination of paclitaxel and metabolites in mouse plasma,tissues, urine and faeces by semi-automated reversed-phase high-performance liquid chromatography

We have developed and validated a sensitive and selective assay for the quantification of paclitaxel and its metabolites 6α,3′-p-dihydroxypaclitaxel, 3′-p-hydroxypaclitaxel and 6α-hydroxypaclitaxel in plasma, tissue, urine and faeces specimens of mice. Tissue and faeces were homogenized (approximately 0.1–0.2 g/ml) in bovine serum albumin (40 g/I) in water, and urine was diluted (1:5, v/v) in blank human plasma. Sample pretreatment involved liquid-liquid extraction of 200–1000 μl of sample with diethyl ether followed by automated solid-phase extraction using cyano Bond Elut column. 2′-Methylpaclitaxel was used as internal standard. The overall recovery of the sample pretreatment procedure ranged from 76 ot 85%. In plasma, the lower limit of detection (LOD) and the lower limit of quantitation (LLQ) are 15 and 25 ng/ml, respectively, using 200 μl of sample. In tissues, faeces and urine the LLQs are 25–100 ng/g, 125 ng/g and 25 ng/ml, respectively, using 1000 μl (faeces: 200 μl) of homogenized or diluted sample. The concentrations in the various biological matrices, for validation procedures spiked with known amounts of the test compounds, are read from calibration curves constructed in blank human plasma in the range 25–100 000 ng/ml for paclitaxel and 25–500 ng/ml for the metabolites. The accuracy and precision of the assay fall within the generally accepted criteria for bio-analytical assays.     

Sensitive high-performance liquid chromatographic assay for endralazine and two of its metabolites in human plasma

Endralazine (I) is a new antihypertensive which is chemically and pharmacologically related to hydralazine and dihydralazine. A sensitive high-performance liquid chromatographic-fluorescence assay for the drug and two of its metabolites [methyltriazoloendralazine (VII) and hydroxymethyltriazoloendralazine (VIII)] in human plasma was developed. After conversion of I and its internal standard to triazolopyridopyridazine derivatives the latter and metabolites were separated by high-performance liquid chromatography and detected using their fluorescence. The limits of detection of the assay were 1 nmol/l for I and VII and 0.1 nmol/l for VIII. Intra-assay coefficients of variation were 2.5–5.1% for I (range 1000–10 nmol/l), 4.2–4.5% for VII (range 100–5 nmol/l) and 3.4–5.7% for VIII (range 100–1 nmol/l). Following oral administration of 5 and 10 mg of I to two normal volunteers (slow acetylators) peak plasma levels of I occurred between 0.75 and 1 h after the dose, and declined in a biexponential fashion. The terminal half-life ranged from 2.8–3.7 h. These results contrast with those obtained for hydralazine in plasma where in vitro and in vivo half-lives were 30 min.     

Determination of amdinocillin in plasma and urine by high-performance liquid chromatography

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of amdinocillin (formerly mecillinam) in human plasma and urine. The assay is performed by direct injection of a plasma protein-free supernatant or a dilution of urine. A 10-μm μBondapak phenyl column with an eluting solvent of water—methanol—1 M phosphate buffer, pH 7 (70:30:0.5) was used, with UV detection of the effluent at 220 nm. Azidocillin potassium salt [potassium-6-(d-(-)-α-azidophenyacetamido)-penicillanate] was used as the internal standard and quantitation was based on peak height ratio of amdinocillin to that of the internal standard. The assay has a recovery of 74.4 ± 6.3% (S.D.) in the concentration ranges of 0.1–20 μg per 0.2 ml of plasma with a limit of detection equivalent to 0.5 μg/ml plasma. The urine assay was validated over a concentration range of 0.025–5 mg/ml of urine, and has a limit of detection of 0.025 mg/ml (25 μg/ml) using a 0.1-ml urine specimen per assay.The assay was applied to the determination of plasma and urine concentrations of amdinocillin following intravenous administration of a 10 mg/kg dose of amdinocillin to two human subjects. The HPLC and microbiological assays were shown to correlate well for these samples.     

Determination of picotamide in human plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of picotamide in human plasma and urine is described. After addition of an internal standard (bamifylline), the plasma and urine samples were subjected to liquid—liquid extraction and clean-up procedures. The final extracts were evaporated to dryness and the resulting residues were reconstituted in 100 μl of methanol—water (50:50, v/v) and chromatographed on a LiChrosorb RP-SELECT B reversed-phase column coupled to an ultraviolet detector monitored at 230 nm. Chromatographic analysis takes about 10 min per sample. The assay was linear over a wide range and has a limit of detection of 0.005 and 0.1 μg/ml in plasma and urine, respectively. It was selective for picotamide, accurate and robust and thus suitable for routine assays after therapeutic doses of picotamide.     

Gas chromatographic determination of novel valproyl taurinamide derivatives in mouse and dog plasma

Valproyl taurinamides are a novel group of compounds that possess anticonvulsant activity. In this study a gas chromatographic micromethod was developed for the quantification of selected valproyl taurinamides and some of their metabolites in biological samples. Valproyl taurinamide (VTD), N-methyl valproyl taurinamide (M-VTD), N,N-dimethyl valproyl taurinamide (DM-VTD) and N-isopropyl valproyl taurinamide (I-VTD) were analyzed in mouse and dog plasma and in dog urine using gas chromatography. Flame ionization detection and mass spectrometric detection were compared. The plasma samples were prepared by solid-phase extraction using C(18) cartridges. The urine samples were prepared by liquid-liquid extraction. The sample volume used was 100 microl of dog plasma, 50 microl of mouse plasma and 20 microl of dog or mouse urine. The quantification range of the method was 1.5-50 mg/l in dog plasma (VTD only), 2.5-250 mg/l in mouse plasma (0.7-90 pmol injected) and 0.04-2 mg/ml in dog urine (VTD only). The inter-day precision in plasma and urine samples was around 10% for all quantified concentrations except LOQ (15-20%). The accuracy for all four compounds was between 90 and 110% within the entire concentration range. The developed method was suitable for quantification of a series of CNS-active valproyl taurineamide derivatives in biological samples at relevant in vivo concentrations.     

Design,synthesis and biological evaluation of selected 3-[3-(amino) propoxy] benzenamines as acetylcholinesterase inhibitors

The present paper describes design, synthesis, and biological evaluation of a series of some 3-[3-(amino)propoxy]benzenamines as acetylcholinesterase inhibitors using mice as a model and piracetam as a reference drug. The structures of these compounds were confirmed by spectral analysis and compounds were tested for memory enhancing activity using elevated plus maze test and acetylcholinesterase inhibitory assay. The inhibitory range of synthesized compounds was from 8.99 to 28.31 μM. The synthesized compounds possessed higher or equivalent percent retention as compared to piracetam at 1 mg/kg with no other CNS-related activities (locomotor and muscle relaxant, analgesic and anticonvulsant activities). Compound 3-[3-(imidazolo)propoxy]benzenamine has shown significant dose-dependent (1 and 3 mg/kg) memory enhancing activity, while 3-[3-(pyrrolidino)propoxy]benzenamine also showed activity equivalent to reference drug piracetam at 1 mg/kg. Both compounds 3-[3-(pyrrolidino)propoxy]benzenamine and 3-[3-(imidazolo)propoxy]benzenamine were also found to show AChE inhibition with IC₅₀ value of 8.99 and 17.87 μM. The molecular docking, MM-GBSA and molecular dynamics simulation studies were performed in order to establish a relationship between the biological results. RMSD, root-mean-square fluctuations, and interaction patterns of 10a–AChE and Sck–AChE complexes proved that the binding affinity of 10a toward AChE was highly stable with the proposed binding orientations.     

The determination of allopurinol and oxipurinol in human plasma and urine

A method is described for allopurinol and oxipurinol assay within human plasma and urine in the range expected during therapy. The method is based on high-performance ion-exchange chromatography following an efficient sample purification step using Chelex-100 resin in the Cu²⁺ form. Linear calibration curves are produced for allopurinol over the range 0.05–10 μmole/1 (0.068–1.36 μg/ml) in plasma and 0.005–1 mmole/1 (0.68–136 μg/ml) in urine and for oxipurinol 0.5–100 μmole/1 (0.076–15.2μg/ml) in plasma and 0.1–2 mmole/1 (15.2–304 μg/ml) in urine.     

High-performance liquid chromatographic determination of tazobactam and piperacillin in human plasma and urine

A high-performance liquid chromatographic (HPLC) method with ultraviolet (UV) absorbance was developed for the analysis of piperacillin-tazobactam (tazocillin), in plasma and urine. The detection was performed at 218 nm for tazobactam and 222 nm for piperacillin. The procedure for assay of these two compounds in plasma and of piperacillin in urine involves the addition of an internal standard (ceftazidime for tazobactam and benzylpenicillin for piperacillin) followed by a treatment of the samples with acetonitrile and chloroform. To quantify tazobactam in urine, diluted samples were analysed using a column-switching technique without internal standard. The HPLC column, LiChrosorb RP-select B, was equilibrated with an eluent mixture composed of acetonitrile-ammonium acetate (pH 5). The proposed technique is reproducible, selective, and reliable. The method has been validated, and stability tests under various conditions have been performed. Linear detector responses were observed for the calibration curve standards in the ranges 5–60 μg/ml for tazobactam, and 1–100 μg/ml for piperacillin and spans what is currently though to be the clinically relevant range for tazocillin concentrations in body fluids. The limit of quantification was 3 μg/ml for tazobactam and 0.5 μg/ml for piperacillin in plasma and urine. Extraction recoveries from plasma proved to be more than 85%. Precision, expressed as C.V., was in the range 0.4–18%.     

Automated analysis of a novel anti-epileptic compound, CGP 33 101, and its metabolite, CGP 47 292, in body fluids by high-performance liquid chromatography and liquid-solid extraction

Automated procedures for the determination of CGP 33 101 in plasma and the simultaneous determination of CGP 33 101 and its carboxylic acid metabolite, CGP 47 292, in urine are described. Plasma was diluted with water and urine with a pH 2 buffer prior to extraction. The compounds were automatically extracted on reversed-phase extraction columns and injected onto an HPLC system by the automatic sample preparation with extraction columns (ASPEC) automate. A Supelcosil LC-18 (5 μm) column was used for chromatography. The mobile phase was a mixture of an aqueous solution of potassium dihydrogen phosphate, acetonitrile and methanol for the assay in plasma, and of an aqueous solution of tetrabutylammonium hydrogen sulfate, tripotassium phosphate and phosphoric acid and of acetonitrile for the assay in urine. The compounds were detected at 230 nm. The limit of quantitation was 0.11 μml/l (25 ng/mol) for the assay of CGP 33 101 in plasma, 11 μmol/l (2.5 μg/ml) for its assay in urine and 21 μmol/l (5 μg/ml) for the assay of CGP 47 292 in urine.     

Determination of a chemoprotective agent, 2-(allylthio)pyrazine, in plasma, urine and tissue homogenates by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the determination of a chemoprotective agent, 2-(allylthio)pyrazine (I), in human plasma and urine, and in rat blood and tissue homogenate using diazepam as an internal standard. The sample preparation was simple; 2.5 volumes of acetonitrile were added to the biological sample to deproteinize it. A 50–100 μl aliquot of the supernatant was injected onto a C₁₈ reversed-phase column. The mobile phase employed was acetonitrile–water (55:45, v/v), and it was run at a flow-rate of 1.5 ml/min. The column effluent was monitored using an ultraviolet detector at 330 nm. The retention times for I and the internal standard were 4.0 and 5.1 min, respectively. The detection limits of I in human plasma and urine, and in rat tissue homogenate (including blood) were 20, 20 and 50 ng/ml, respectively. The coefficients of variation of the assay (within-day and between-day) were generally low (below 6.1%) in a concentration range from 0.02 to 10 μg/ml for human plasma and urine, and for rat tissue homogenate. No interferences from endogenous substances were found.     

Simultaneous determination of quinine and four metabolites in plasma and urine by high-performance liquid chromatography

The determination of quinine, (3S)-3-hydroxyquinine, 2′-quininone and (10R)- and (10S)-10,11-dihydroxydihydroquinine in plasma and urine samples is described. This is the first time the R and S configurations have been correctly assigned to the two metabolites of 10,11-dihydroxyquinine. One hundred microliter-plasma samples were protein precipitated with 200 μl cold methanol. Urine samples were 10–100× diluted and then directly injected into the HPLC. A reversed-phase liquid chromatography system with fluorescence detection and a Zorbax Eclipse XDB phenyl column and gradient elution was used. The within and between assay coefficients of variation of the method for quinine and its metabolites in plasma and urine was less than 13%. The lower limit of quantitation was in the range of 0.024–0.081 μM.     

Efficient high-performance liquid chromatographic assay for the simultaneous determination of metoprolol and two main metabolites in human urine by solid-phase extraction and fluorescence detection

An improved, more efficient method for the determination of metoprolol and its two metabolites in human urine is reported. The simultaneous analysis of the zwitterionic metoprolol acidic metabolite (III, H117/04) with the basic metabolites α-hydroxymetoprolol (II, H119/66), metoprolol (I) and guanoxan (IV, internal standard) was achieved employing solid-phase extraction and isocratic reversed-phase HPLC. The analytes were extracted from urine (100 μl) using C₁₈ solid-phase extraction cartridges (100 mg), and eluted with aqueous acetic acid (0.1%, v/v)–methanol mixture (40:60, v/v, 1.2 ml). The eluents were concentrated (250 μl) under vacuum, and aliquots (100 μl) were analysed by HPLC with fluorescence detection at 229 nm (excitation) and 309 nm (emission) using simple isocratic reversed-phase HPLC (Novapak C₁₈ radial compression cartridge, 4 μm, 100×5 mm I.D.). Acetonitrile–methanol–TEA/phosphate buffer pH 3.0 (9:1:90, v/v) was employed as the eluent (1.4 ml/min). All components were fully resolved within 18 min, and the calibration curves for the individual analytes were linear (r²≥0.996) within the concentration range of 0.25–40.0 mg/ml. Recoveries for all four analytes were greater than 76% (n=4). The assay method was validated with intra-day and inter-day variations less than 2.5%.     

Simple and selective high-performance liquid chromatographic method for estimating plasma quinidine levels

A reversed-phase, high-performance liquid chromatographic method employing fluorescence detection is described for the rapid quantification of plasma levels of quinidine, dihydroquinidine and 3-hydroxyquinidine. It involves protein precipitation with acetonitrile followed by direct injection of the supernatant into the chromatograph. For the preparation of plasma standards, pure 3-hydroxyquinidine was isolated from human urine by a simplified thin-layer chromatographic procedure. The mobile phase for the chromatography was a mixture of 1.5 mM aqueous phosphoric acid and acetonitrile (90:10) at a flow-rate of 2 ml/min. The intra-assay coefficient of variation for the assay of quinidine and 3-hydroxyquinidine over the concentration range 2.5–20 μmole/l was < 1% for both. Interassay coefficients of variation for quinidine (10 μmole/l) and 3-hydroxyquinidine (5 μmole/l) were 3.5% and 4.0% with detection limits of 50 and 25 μmole/l respectively. The method correlated well (r² = 0.96) with an independently developed gas—liquid chromatographic—nitrogen detection assay for quinidine which also possessed a high degree of precision. (Intra-assay coefficient of variation 3.6% at 20 μmole/l). As expected, comparison of the high-performance liquid chromatographic assay with a published protein precipitation—fluorescence assay showed poor correlation (r² = 0.78).     

Quantitation of itraconazole in rat heparinized plasma by liquid chromatography–mass spectrometry

A liquid chromatographic–mass spectrometric (LC–MS) assay was developed and validated for the determination of itraconazole (ITZ) in rat heparinized plasma using reversed-phase HPLC combined with positive atmospheric pressure ionization (API) mass spectrometry. After protein precipitation of plasma samples (0.1 ml) with acetonitrile containing nefazodone as an internal standard (I.S.), a 50-μl aliquot of the supernatant was mixed with 100 μl of 10 mM ammonium formate (pH 4.0). An aliquot of 25 μl of the mixture was injected onto a BDS Hypersil C₁₈ column (50×2 mm; 3 μm) at a flow-rate of 0.3 ml/min. The mobile phase comprising of 10 mM ammonium formate (pH 4) and acetonitrile (60:40, v/v) was used in an isocratic condition, and ITZ was detected in single ion monitoring (SIM) mode. Standard curves were linear (r²≥0.994) over the concentration range of 4–1000 ng/ml. The mean predicted concentrations of the quality control (QC) samples deviated by less than 10% from the corresponding nominal values; the intra-assay and inter-assay precision of the assay were within 8% relative standard deviation. Both ITZ and I.S. were stable in the injection solvent at room temperature for at least 24 h. The extraction recovery of ITZ was 96%. The validated assay was applied to a pharmacokinetic study of ITZ in rats following administration of a single dose of itraconazole (15 mg/kg).     

Absorption and metabolism of oral progesterone.

The absorption, metabolism, and clearance of progesterone from the peripheral circulation were investigated in five postmenopausal women after oral administration of 100 mg daily for five consecutive days. Maximal plasma concentrations of progesterone were observed within four hours after ingestion of the last dose, when the range (22.11-34.18 nmol/l; 696-1077 ng/100 ml) was comparable with that observed during the mid-luteal phase of the ovarian cycle. The surge in values lasted six hours, and progesterone concentrations remained raised for at least 96 hours. Of the three metabolites studied, the plasma concentrations of pregnanediol-3 alpha-glucuronide were most raised by treatment, the peak values ranging from 1097 nmol/l (54.9 microgram/100 ml) to over 2000 nmol/l (100 microgram/100 ml), which was the upper limit of the assay used. Concentrations of 17-hydroxyprogesterone were least raised, and the peak values ranged from 4.32 to 9.68 nmol/l (143-319 ng/100 ml). The plasma profile of 20 alpha-dihydroprogesterone most closely approximated that of progesterone, although the range of maximal values was lower (7.11-16.06 nmol/l; 228-514 ng/100 ml). Plasma concentrations of oestradiol were unchanged by giving progesterone. It is concluded that the increases in circulating concentrations of progesterone and the biologically active metabolite 20 alpha-dihydroprogesterone, and the duration of these increases, were sufficient to modulate the biochemistry of responsive tissues. Oral progesterone may thus have a therapeutic role, and this route of administration merits further investigation.     

Spectrofluorimetric determination of nomifensine in human plasma and urine by derivatization with fluorescamine

A sensitive, simple and rapid spectrofluorimetric method was developed for the determination of nomifensine in human plasma and urine. The present method was based on the derivatization by fluorescamine in phosphate buffer at pH 4.0 to produce a highly fluorescent product which was measured at 488 nm (excitation at 339 nm). The method was validated according to the ICH guidelines with respect to linearity, limit of detection, limit of quantification, accuracy, precision, recovery and robustness. The assay was linear over the concentration ranges 100–2,000 and 50–2,000 ng/mL for plasma and urine, respectively. The limits of detection were calculated to be 13.9 and 7.5 ng/mL for plasma and urine, respectively. The method was successfully applied to the analysis of the drug in human plasma and urine. Copyright © 2011 John Wiley & Sons, Ltd.     

Simultaneous quantitative analysis of methyl salicylate,ethyl salicylate and salicylic acid from biological fluids using gas chromatography–mass spectrometry

A gas chromatographic–mass spectrometric (GC–MS) assay was developed for the quantitative analysis of methyl salicylate (MeS), ethyl salicylate (ES) and salicylic acid (SA) from biological fluids. The method was validated from 100-μl rat liver homogenate preparations (5 mg/ml protein) in 70 mM KH₂PO₄ (pH 7.4) buffer and from 100 μl rat plasma. The samples were extracted with chloroform, derivatized with BSTFA and quantitated by GC–MS in the SIM mode. The standard curves ranged from 31 ng/ml to 800 or 1250 ng/ml. Relative standard deviations and bias were less than 11% in plasma and homogenate for all compounds except SA which evidenced greater variability. The assay was used in preliminary experiments to characterize the pharmacokinetics of MeS in rats.     

Ultrasensitive assay for three polyphenols (catechin,quercetin and resveratrol) and their conjugates in biological fluids utilizing gas chromatography with mass selective detection

The concentrations of three polyphenols ((+)-catechin, quercetin and trans-resveratrol) in blood serum, plasma and urine, as well as whole blood, have been measured after their oral and intragastric administration, respectively, to humans and rats. The method developed for this purpose utilized ethyl acetate extraction of 100 μl samples and their derivatization with bis(trimethylsilyl)trifluoroacetamide (BSTFA) followed by gas-chromatographic analysis on a DB-5 column followed by mass selective detection employing two target ions and one qualifier ion for each compound. Total run time was 17 min with excellent resolution and linearity. The limits of detection (LOD) and quantitation (LOQ) were an order of magnitude less than for any previously published method, being 0.01 μg/l and 0.1 μg/l, respectively, for all compounds. Recovery at 1 μg/l and 10 μg/l was >80% in all instances but one, and was >90% in 50%. Imprecision was acceptable at 0.25 and 1.0 μg/l, concentrations below the LOQ of previous methods. Aglycones released from conjugates after hydrolysis were easily measurable. Optimal conditions for hydrolysis were established. After oral administration of the three polyphenols to humans, their conjugates vastly exceeded the concentrations of the aglycones in both plasma and urine. Concentrations peaked within 0.5–1.0 h in plasma and within 8 h in urine. During the first 24 h, 5.1% of the (+)-catechin and 24.6% of the trans-resveratrol given were recovered in the urine (free plus conjugated). This method can be proposed as the method of choice to assay these polyphenols and their conjugates in biological fluids.     

Simultaneous determination of a new anticancer agent (NB-506) and its active metabolite in human plasma and urine by high-performance liquid chromatography with ultraviolet detection

A high-performance liquid chromatographic method with ultraviolet detection has been developed to quantify NB-506 and its active metabolite in human plasma and urine. This method is based on solid-phase extraction, thereby allowing the simultaneous measurement of the drug and metabolite with the limit of quantification of 0.01 μg/ml in plasma and 0.1 μg/ml in urine. Standard curves for the compounds were linear in the concentration ranges investigated. The range for the drug in plasma was 0.01–2.5 μg/ml, and for the metabolite 0.01–1 μg/ml. In urine, the range for both compounds was 0.1–10 μg/ml. The method was validated and applied to the assay of plasma and urinary samples from phase I studies.