Determination of propofol in rat whole blood and plasma by high-performance liquid chromatography

A simple, accurate and sensitive high-performance liquid chromatographic method was developed for the determination of propofol, an intravenous anaesthetic agent, in rat whole blood or plasma samples. The method is based on precipitation of the protein in the biological fluid sample and direct injection of the supernatant into an HPLC system involving a C₁₈ reversed-phase column using a methanol-water (70:30) mobile phase delivered at 1 ml/min. Propofol and the internal standard (4-tert.-octylphenol) were quantified using a fluorescence detector set at 276 nm (excitation) and 310 nm (emission). The analyte and internal standard had retention times of 6.3 and 10.5 min, respectively. The limit of quantification for propofol was 50 ng/ml using 100 μl of whole blood or plasma sample. Calibration curves were linear (r²=0.99) over a 1–10 μg/ml concentration range and intra- and inter-day precision were between 4–11%. The assay was applied to the determination of propofol whole blood pharmacokinetics and propofol whole blood to plasma distribution ratios in rats.     

Flame atomic emission spectrometric determination of aluminium in a bovine blood plasma matrix

A flame atomic emission spectrometric method, is described for the determination of aluminium in bovine blood plasma matrices. Plasma samples are wet-digested and solutions are aspirated into a conventional nitrous oxide-acetylene flame. Analyte emission is monitored at 396.15 nm with corrections for background emission being obtained from measurements several tenths nm on both sides of the aluminium line. The mean recovery of 0.3–5 μg/ml aluminium added to model solutions containing 500–5000 μg Na/ml, 50–1000 μg Ca/ml, 2000–5000 μg K/ml, or simulated plasma digests containing Na, K, and Ca was 100,6% (SD = 10.9, df = 60); the mean recovery of 0.3, 0.5, and 1.0 μg/ml aluminium added to blood plasma before digestion was 94.3% (SD = 9.8, df = 33) indicating no serious interferences. For standard solutions, the detection limit (signal: peak-to-peak noise = 1) was 0.02 μg/ml by flame emission, and 0.12 μg/ml by atomic absorption measurements with the same instrument. A sample taken through the analytical procedure, gave a detection limit of 0.05 μg/ml suggesting the submicrogram per milliliter region as the lower practical limit of the method.     

Analysis of methanol or formic acid in body fluids by headspace solid-phase microextraction and capillary gas chromatography

Methanol and its metabolite formic acid have been found extractable from human whole blood and urine by headspace solid-phase microextraction (SPME) with a Carboxen/polydimethylsiloxane fiber. The headspace SPME for formic acid was carried out after derivatization to methyl formate under acidic conditions. The determinations of both compounds were made by using acetonitrile as internal standard (IS) and capillary gas chromatography (GC) with flame ionization detection. The headspace SPME–GC gave sharp peaks for methanol, methyl formate and I.S.; and low background noises for whole blood and urine samples. Extraction efficiencies were 0.25–1.05% of methanol and 0.38–0.84% formic acid for whole blood and urine. The calibration curves for methanol and formic acid showed excellent linearity in the range of 1.56 to 800 and 1.56 to 500 μg/0.5 ml of whole blood or urine, respectively. The detection limits were 0.1–0.5 μg/0.5 ml for methanol and 0.6 μg/0.5 ml for formic acid for both body fluids. The within-day relative standard deviations in terms of extraction efficiency for both compounds in whole blood and urine samples were not greater than 9.8%. By using the established SPME method, methanol and formic acid were successfully separated and determined in rat blood after oral administration of methanol.     

Chiral Assay of Atenolol Present in Microdialysis and Plasma Samples of Rats Using Chiral CBH as Stationary Phase

Two different enantioselective chiral chromatographic methods were developed and validated to investigate the disposition of the β₁-receptor antagonist atenolol in blood and in brain extracellular fluid of rats (tissue dialysates). System A for the plasma samples was a one-column chromatographic system with a Chiral CBH column with an aqueous buffer as mobile phase into which cellobiose was added for selective regulation of the retention of the internal standard, (S)-metoprolol. The plasma samples were analysed after a simple extraction procedure. The limit of quantitation was 0.2 μg/ml for the atenolol enantiomers. The repeatability of the medium concentration quality control plasma sample (6.0 μg rac-atenolol/ml) was 11–18% for the enantiomers. The dynamic linear range of the plasma samples was 0.5–20 μg/ml. For system B, since atenolol is an extremely hydrophilic drug, the tissue dialysate sample required a much more sensitive system as compared to the plasma samples. A coupled column system was used for peak compression of the enantiomers in the eluate after the separation on the Chiral CBH column, hence increasing the detection sensitivity. The limit of quantification was 0.045 μg/ml for the atenolol enantiomers in artificial CSF. The repeatability of the medium concentration quality control samples (0.1 and 4.0 μg rac-atenolol/ml in artificial CSF and Hepes Ringer, respectively) was 2.8–9.3% for the two enantiomers. The dynamic linear range of the brain samples was 0.05–1.0 and 0.5–20 μg/ml in artificial CSF and Hepes Ringer, respectively. Chirality 9:329–334, 1997. © 1997 Wiley-Liss, Inc.     

Simultaneous determination of amlodipine and metoprolol in their combined dosage form using a synchronous fluorescence spectrofluorimetric method

Highly sensitive, rapid, accurate and precise synchronous fluorescence spectrofluorimetric method has been developed for simultaneous analysis of a mixture of amlodipine (AMD) and metoprolol (MET). The method relies on measuring the relative synchronous fluorescence intensity of both drugs at Δλ of 90 nm in acetate buffer solution at pH 5. The experimental parameters influencing the developed method were investigated and optimized. The method was linear over the ranges 0.2–2 μg/ml and 0.5–10 μg/ml for AMD and MET, respectively. The limits of detection were 50 ng/ml for AMD and 130 ng/ml for MET while the limits of quantitation were 150 ng/ml for AMD and 390 ng/ml for MET. The developed method was applied successfully for the determination of the two drugs in their co‐formulated tablet. The mean percent recoveries were found to be 100.51 and 99.57 for AMD and MET, respectively.     

4-Dimethylaminobenzylamine as a sensitive chemiluminescence derivatization reagent for 5-hydroxyindoles and its application to their quantification in human platelet-poor plasma

A selective and sensitive high-performance liquid chromatographic method with chemiluminescence detection for the determination of 5-hydroxyindoles is described, based on the reaction of 5-hydroxyindoles with 4-dimethylaminobenzylamine. Serotonin, 5-hydroxyindole-3-acetic acid, 5-hydroxytryptophol, 5-hydroxyindole-3-acetamide and N-acetyl-5-hydroxytryptamine were used as model compounds to optimize the derivatization and chemiluminescent reaction. The reagent reacts with 5-hydroxyindoles in slightly alkaline media in the presence of potassium hexacyanoferrate(III) to give the corresponding derivatives, which can be separated on a reversed-phase column, Wakosil-II 5C18RS, with aqueous acetonitrile as an eluent. The derivatives were detected by peroxyoxalate chemiluminescence detection. The detection limits are in the range of 0.5–1.2 fmol per 100-μl injection. The method was applied to the simultaneous determination of serotonin and 5-hydroxyindole-3-acetic acid in human platelet-poor plasma.     

High-performance liquid chromatographic determination of hydrocortisone and methylprednisolone and their hemisuccinate esters in human serum

A high-performance liquid chromatographic method is described for the simultaneous determination of methylprednisolone (MP) and methylprednisolone hemisuccinate (MPHS), or hydrocortisone (HC) and hydrocortisone hemisuccinate (HCHS) in human serum. Reversed-phase liquid chromatography was performed on a microparticulate C₁₈ column (Spherisorb, 5 μm) using a mobile phase of 2% glacial acetic acid, 30–35% acetonitrile, 70–65% water with ultraviolet detection (254 nm). The method uses 17α-hydroxyprogesterone as the internal standard for the determination of methylprednisolone and its hemisuccinate ester, or 11-deoxy-17-hydroxycorticosterone as the internal standard for the determination of hydrocortisone and its hemisuccinate ester. The sensitivity is 0.03 μg/ml for HC, 0.07 μg/ml for MP, 0.04 μg/ml for MPHS, and 0.10 μg/ml for HCHS, with a detection limit of 0.02 μg/ml for all four steroids. Calibration curves are linear up to 3 μg/ml for MP or MPHS (as equivalent MP) and up to 4 μg/ml for HC and 7 μg/ml (as equivalent HC) for HCHS. The pooled relative standard deviation for replicate samples for each steroid is < 7%. Plasma concentration—time curves are reported for MP and MPHS or HC and HCHS of two human subjects following intramuscular administration of 125 mg of methylprednisolone sodium succinate for injection, U.S.P., or 250 mg of hydrocortisone sodium succinate for injection, U.S.P.     

Simultaneous determination of phenol,cresol, xylenol isomers and napthols in urine by capillary gas chromatography

An attempt was made to establish a method for the simultaneous determination of urinary concentrations of phenol, o-, p- and m-cresols, 1 and 2-naphthol and xylenol isomers by capillary gas chromatography. Urine samples were extracted after acid hydrolysis of glucuronides and sulfates by solid-phase extraction. The ten substances were separated gas chromatographically using a capillary column (Ultra 2) of cross-linked 5% phenylmethyl silicone. Calibration graphs were linear for 5–100 μg/ml of all the phenols determined. The corresponding detection limits for phenolic compounds varied from 0.1 to 0.2 μg/ml. The relative standard deviations for samples in urine were in the range 2.6–16.6% and the accuracy was in the range 1.4–25%. Recoveries were generally over 80%.     

Determination of N-(trans-4-isopropylcyclohexylcarbonyl)-d-phenylalanine in human plasma by solid-phase extraction and column-switching high-performance liquid chromatography with ultraviolet detection

A column-switching high-performance liquid chromatography method with ultraviolet detection at 210 nm has been developed for the determination of N-(trans-4-isopropylcyclohexylcarbonyl)-d-phenylalanine (AY4166, I) in human plasma. Plasma samples were prepared by solid-phase extraction with Sep-Pak Light tC₁₈, followed by HPLC. The calibration graph for I was linear in the range 0.1–20 μg/ml. The limit of quantitation of I, in plasma, was 0.05 μg/ml. The recovery of spiked I (0.5 μg/ml) to drug-free plasma was over 92% and the relative standard deviation of spiked I (0.5 μg/ml) compared to drug-free plasma was 4.3% (n = 8).     

Spectrofluorimetric study on fluorescence quenching of tyrosine and l‐tryptophan by the aniracetam cognition enhancer drug: quenching mechanism using Stern–Volmer and double‐log plots

A novel approach on fluorescence quenching of tyrosine and l ‐tryptophan is presented for spectrofluorimetric determination of aniracetam in drug substances and products. The quenching mechanism was investigated using Stern–Volmer plots and ultraviolet spectra figures of quencher–fluorophore mixtures. Binding constant and stoichiometry were calculated using double‐log plots. The spectrofluorimetric method was optimized for the experimental conditions affecting fluorescence quenching including fluorophore concentration, diluent, and reaction time. Moreover, the pH‐rate profile of aniracetam was studied using simple kinetics and found to be stable within the pH range 5–8. Fluorescence quenching of tyrosine and l ‐tryptophan were observed on addition of aniracetam in aqueous medium at pH 5.5–6.5. Aniracetam quenched the fluorescence of tyrosine and l ‐tryptophan in the concentration range 1–20 μg/ml and 0.3–20 μg/ml, respectively, with binomial relationships between quenching values (ΔF) and aniracetam concentration. Limits of detection were found to be 0.10 μg/ml for tyrosine–aniracetam and 0.14 μg/ml for l ‐tryptophan–aniracetam. Method validation was performed as per ICH guidelines and demonstrated that the developed spectrofluorimetric method was accurate, precise, specific, and suitable for analysis of aniracetam in routine quality control laboratories. All experimental materials and solvents used are eco‐friendly, indicating that the cited spectrofluorimetric procedure is an excellent green method.     

In vivo stimulation of ovarian development in the red swamp crayfish,Procambarus clarkii (Girard), by 5-hydroxytryptamine

Summary

The possibility that biogenic amines affect ovarian development in the red swamp crayfish, Procambarus clarkii, was investigated. Females were administered 15 μg/g body weight (bw) of norepinephrine, dopamine, 5-hydroxytryptamine (5-HT) or octopamine on days 1, 5 and 10 and were sacrificed on day 15. Crayfish given 5-HT showed significant increases in ovarian index (30.5%) and oocyte size (34.0%) over the concurrent controls, while norepinephrine, dopamine and octopamine did not significantly affect either the ovarian index or oocyte size. Significantly more labeling by ¹⁴C-leucine of ovarian proteins was found in ovaries of crayfish that were injected with 5-HT in vivo, but when ovarian lobes from crayfish that had not been injected with 5-HT were incubated in vitro with 5-HT added to the incubation medium, no significant change in the level of incorporation of ¹⁴C-leucine into ovarian proteins occurred.

The 5-HT receptor blocker LY53857 (25 μg/g bw) retarded ovarian development. The 5-HT releaser fenfluramine and the 5-HT potentiator fluoxetine (both 15 μg/g bw) were also used. Crayfish given fenfluramine, fluoxetine, fenfluramine plus 5-HT or fluoxetine plus 5-HT showed significant increases of ovarian index (24.0–102.8%), oocyte size (20.0–87.4%) and in vitro ¹⁴C-leucine labeling of ovarian proteins (30.6–123.6%) over the concurrent controls. The ovaries of crayfish that received the 5-hydroxytryptaminergic neurotoxin 5,6-dihydroxytryptamme (10 μg/g bw) did not show any significant change as compared with the initial control. These findings are consistent with the hypothesis that 5-HT, which is present in the central nervous system of Procambarus clarkii, exerts its stimulatory effect on the ovary of this crayfish indirectly by triggering release of the ovary-stimulating hormone.     

In vitro motor activity of the lamprey (Lampetra japonica) intestine

The intestine of the lamprey (primitive vertebrate), was divided into four segments (SI-SIV) of about 4cm from oral to anal. The frequency and amplitude of their spontaneous motor activities (intraluminal pressure) were studied in vitro. Frequency gradient from oral SI (1.44 cycle/min) to anal SIV (0.26 cycle/min) was found. In contrast, the amplitude gradient (0.14–0.36 mmHg) was quite the reverse and SIV was the largest and SI was the smallest. Raising the temperature from 5 to 10–15°C almost linearly increased both frequency and amplitude in Arrhenius's plot. Acetylcholine (1–10 mM) and 5-HT (1–10 μM) directly induced the contractions of the longitudinal and circular smooth muscles in the presence of 1 μg/ml tetrodotoxin (TTX). Atropine (5–20 μM) shifted the dose-response curves of acetylcholine to the right but hardly affected the 5-HT responses. Spiperone and ketanserine (10–40 μM) shifted the dose-response curves of 5-HT to the right but hardly affected the acetylcholine responses. Stimulating the anal part of SIII and SIV by 3–9 g balance weights via hook and pulley induced the contractions (0.2–2 g) of both oral and anal site (26.5% of trial) or one side of oral (50.0%) or anal site (23.5%) about 2–6 mm apart from the stimulated point. Intrinsic reflex arcs seem to operate in the caudal lamprey intestine. The intrinsic reflex responses were suppressed by about 50% in the presence of 14–29 μg/ml curare and suppressed by 80–100% in the presence of 20 μM spiperone or ketanserine. Atropine 20 μM had almost no inhibitory effects on the reflex. It was suggested that lamprey intestinal smooth muscles seem to have separate receptors for acetylcholine and 5-HT. Intrinsic reflex may be mediated both by cholinergic and serotonergic interneurones and more probably by serotonergic motoneurones. These characteristics of the lamprey were discussed by comparison with those of other vertebrate intestines.     

Variation in the sensitivity to metalaxyl of Pythium spp. isolated from carrot and other sources

Over a 3-yr period 261 isolates of 17 species of Pythium were tested for sensitivity to metalaxyl at concentrations of 5, 50 or 100 μ/ml. A wide range of responses was observed, from isolates where growth ceased at 5 μg/ml to those where growth at 100 μg/ml was similar to that of the untreated controls. In further tests isolates of 11 different species had ED50's < 1 μg/ml. A lower sensitivity was detected in isolates of six Pythium spp. where values in the range 1–10 μg/ml were obtained. This lower sensitivity was not related to previous known use of metalaxyl. Three isolates of Pythium dissotocum from sites where the fungicide had been used repeatedly had ED50's > 100 μg/ml and were considered resistant. The resistance was stable over a 2-yr period and isolates were cross-resistant to furalaxyl, benalaxyl, ofurace, cyprofuram and oxadixyl. Increasing concentrations of metalaxyl reduced or prevented the production of zoospores by four species of Pythium, although when zoospores were produced, this was followed by the normal processes of encystment and germination. Culturing P. dissotocum on different sub-lethal concentrations of metalaxyl for 18 wk did not induce a high level of resistance to the fungicide.     

Manual and automated (robotic) high-performance liquid chromatography methods for the determination of mycophenolic acid and its glucuronide conjugate in human plasma

A manual and an automated (Zymark PyTechnology robot) HPLC method for simultaneous determination of plasma mycophenolic acid (MPA) and its glucuronide conjugate (MPAG) are described here. Both methods are reproducible and accurate, and both are equivalent in all respects, including quantification limits (MPA, 0.100 μg/ml; MPAG, 4.00 μg/ml), range (using 0.05–0.5 ml of plasma: MPA, 0.0500–20.0 μg/aliquot; MPAG, 2.00–200 μg/aliquot), precision, and accuracy. MPA and MPAG were stable under the conditions used with both methods. Results from aliquots of paired control samples, analyzed by the manual method over three years at six analytical laboratories, showed excellent agreement in precision and accuracy.     

In Vitro Antibiotic Susceptibilities of Borrelia Isolates from Erythema Migrans Lesion of Lyme Disease Patients in Japan

Antibiotic susceptibilities of twelve borrelial isolates from skin of patients with erythema migrans (EM) and ticks (Ixodes persulcatus and I. ovatus) in Japan were examined by in vitro microdilution MIC method and macrodilution MBC method. Nine EM isolates and 3 tick isolates were susceptible to amoxicillin, erythromycin, and minocycline. MICs for Japanese isolates were 0.038–0.30 μg/ml, < 0.012 μg/ml, and < 0.012–0.05 μg/ml, respectively. MBCs were as follows: 0.038–0.88 μg/ml, < 0.012–0.10 μg/ml, and <0.025–0.78 μg/ml, respectively. These antibiotics could be recommended for treatment of patients in early stage of Lyme disease in Japan.     

Simultaneous determination of primidone and its three major metabolites in rat urine by high-performance liquid chromatography using solid-phase extraction

A new high-performance liquid chromatographic method for simultaneous determination of primidone (PRM) and of its three major metabolites, phenobarbital (PB), p-hydroxyphenobarbital (p-HO-PB) and phenylethylmalonamide (PEMA), in rat urine, was developed. After acid hydrolysis, these compounds were extracted from urine by means of a Bond Elut Certify LRC column with good clean-up. The extracts were chromatographed on a C₁₈ reversed-phase column using isocratic elution at 40°C, with UV detection at 227 nm. The limit of detection was 0.5 mg/ml for the four compounds. Good linearity (r²>0.99) was observed within the calibration ranges studied: 37.4–299.3 μg/ml for PRM, 26.4–211.2 μg/ml for PB, 12.5–100.2 μg/ml for p-HO-PB and 12.1–97.0 μg/ml for PEMA. Repeatability was in the range 3.1–6.8%. This method constitutes a useful tool for studies on the influence of various parameters on primidone metabolism.     

High-performance liquid chromatographic assay of 5-fluorouracil in human erythrocytes,plasma and whole blood

A HPLC assay method was modified and validated for the determination of 5-fluorouracil in human red blood cells, plasma and whole blood with a two-fold increased sensitivity (detection limit=10 ng/ml). The assay was linear from 25 to 1500 ng/ml and the accuracy ranged from 96.7 to 103.2% at 25 ng/ml, 94.8 to 99.4% at 500 ng/ml, and 98.9 to 99.5% at 1500 ng/ml. Intra-assay and inter-assay coefficients of variation were less than 8% over the range of concentrations and less than 8% over 10 days of analysis. After intravenous bolus and infusion of 5-fluorouracil in patients with colorectal cancer, the concentrations of 5-fluorouracil in whole blood were 108–111% of plasma concentrations, while packed red blood cells levels were 8–15% of plasma concentrations in the five patients studied. By utilising basic analytical hardware, this represents an accurate, precise, reproducible and affordable method for 5-fluorouracil pharmacokinetics investigation and therapeutic drug monitoring.     

Two spectroscopic methods for estimation of anti-migraine medications: Sumatriptan and Zolmitriptan based on nucleophilic substitution reaction

Sensitive and selective spectrophotometric and spectrofluorimetric methods have been developed for the estimation of two anti-migraine drugs, namely sumatriptan succinate (SUM) and zolmitriptan (ZOL). These methods depend on producing a yellow-coloured product after the reaction of the two drugs with 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl). The reaction products exhibited maximum absorbance at 481 nm in borate buffer of pH 9 and fluorescence emission peak at 540 nm after excitation at 470 nm for the two drugs. The linear ranges were 5–60 μg/ml for SUM and 5–50 μg/ml for ZOL in the spectrophotometric method (Method I), whereas this was 0.4–4 μg/ml for SUM and 0.5–5 μg/ml for ZOL in the spectrofluorimetric method (Method II). The method validity was assessed according to International Council for Harmonisation (ICH) guidelines. Statistical analysis of the results obtained from the proposed and comparison methods confirmed that the proposed methods were highly accurate and precise. The suggested methods could be used for the determination of the mentioned drugs in both pure form and in tablets.     

Determination of N-(trans-4-isopropylcyclohexylcarbonyl)-d-phenylalanine in human plasma by solid-phase extraction and column-switching high-performance liquid chromatography with ultraviolet detection

A column-switching high-performance liquid chromatography method with ultraviolet detection at 210 nm has been developed for the determination of N-(trans-4-isopropylcyclohexylcarbonyl)-d-phenylalanine (AY4166, I) in human plasma. Plasma samples were prepared by solid-phase extraction with Sep-Pak Light tC₁₈, followed by HPLC. The calibration graph for I was linear in the range 0.1–20 μg/ml. The limit of quantitation of I, in plasma, was 0.05 μg/ml. The recovery of spiked I (0.5 μg/ml) to drug-free plasma was over 92% and the relative standard deviation of spiked I (0.5 μg/ml) compared to drug-free plasma was 4.3% (n = 8).     

Chiral HPLC determination and stereoselective pharmacokinetics of tetrahydroberberine enantiomers in rats

Tetrahydroberberine (THB), a racemic mixture of (+)‐ and (?)‐enantiomer, is a biologically active ingredient isolated from a traditional Chinese herb Rhizoma corydalis (yanhusuo). A chiral high performance liquid chromatography method has been developed for the determination of THB enantiomers in rat plasma. The enantioseparation was carried out on a Chiral®‐AD column using methanol:ethanol (80:20, v/v) as the mobile phase at the flow rate 0.4 ml/min. The ultraviolet detection was set at 230 nm. The calibration curves were linear over the range of 0.01–2.5 μg/ml for (+)‐THB and 0.01‐5.0 μg/ml for (?)‐THB, respectively. The lower limit of quantification was 0.01 μg/ml for both (+)‐THB and (?)‐THB. The stereoselective pharmacokinetics of THB enantiomers in rats was studied after oral and intravenous administration at a dose of 50 and 10 mg/kg racemic THB (rac‐THB). The mean plasma levels of (?)‐THB were higher at almost all time points than those of (+)‐THB. (?)‐THB also exhibited greater C_max, and AUC_0–∞, smaller CL and V_d, than its antipode. The (?)/(+)‐enantiomer ratio of AUC_0–∞ after oral and intravenous administration were 2.17 and 1.43, respectively. These results indicated substantial stereoselectivity in the pharmacokinetics of THB enantiomers in rats. Chirality, 2012. © 2012 Wiley Periodicals, Inc.