Determination of carnosine and other biogenic imidazoles in equine plasma by isocratic reversed-phase ion-pair high-performance liquid chromatography.

The isocratic reversed-phase ion-pair high-performance liquid chromatographic technique presented provides a sensitive, rapid and reproducible analytical method for the selective determination of carnosine and other biogenic imidazoles in equine plasma. Plasma was deproteinized with 5-sulphosalicylic acid and the compounds of interest were isolated by sorbent extraction on Bond Elut PRS cartridges. Recoveries were 97-105% and the lowest limits of detection were 58.3-80.1 nM. All compounds of interest were well resolved within a maximum retention time of 9.2 min. The mean equine plasma carnosine level determined by this method was 11.31 microM. Comparative determinations were made in canine and human plasma. Carnosine was not detected in human plasma. Concentrations of imidazole in canine plasma are reported here for the first time.     

Simultaneous Determination of Salicylic Acid and Salicyluric Acid in Plasma and Urine by High Pressure Liquid Chromatography

Abstract

A sensitive, rapid, and specific high pressure liquid chromatographic (HPLC) assay was developed for the determination of salicylic (SA) and salicyluric (SU) acids in plasma and urine. The compounds are extracted into ethyl ether at acid pH, evaporated, and reconstituted prior to instrumental separation. Overall recovery of both compounds is 90 ± 5%, and the sensitivity limits are 150 ng of SU and 300 ng SA per ml of biological fluid. The assay was used for the determination of both compounds in plasma and urine of man following oral doses of 40 mg/kg of sodium salicylate.     

Direct enantiomeric resolution of disopyramide and its metabolite using chiral high-performance liquid chromatography. Application to stereoselective metabolism and pharmacokinetics of racemic disopyramide in man

A method for the simultaneous determination of disopyramide and mono-N-desisopropyldisopyramide enantiomers extracted from human plasma and urine is presented. Separation and quantitation were carried out using two columns coupled in series, and UV detection at 254 nm. First, the racemates of the two compounds were separated using a reversed-phase column, and then the enantiomers were separated using a stereoselective column packed with human alpha 1-acid glycoprotein. The mobile phase was 8 mM phosphate buffer, pH 6.20-2-propanol (92:8, v/v). The coefficients of variation (%) for the plasma daily determination were 6.7% for R(-)- and S(+)-disopyramide at drug levels of 1.5 micrograms/ml, and 8.5% and 7.7% for R(-)- and S(+)-mono-N-desisopropyldisopyramide, respectively, at drug levels of 0.375 micrograms/ml. The method has allowed the study of stereoselective metabolism and pharmacokinetics of disopyramide after oral administration as a racemate.     

Gas chromatographic determination of mefloquine in human and dog plasma using electron-capture detection

A sensitive and selective gas-liquid chromatographic method for the determination of plasma levels of mefloquine in human and dog plasma is described. The drug and internal standard were extracted from plasma at pH 9.0 into isopropyl acetate. After evaporation of the solvent, the residue was taken up in toluene and derivatised with heptafluorobutyrylimidazole. The derivative was quantified by gas-liquid chromatography on a 3% GC GE-SE30 column with electron-capture detection. The limit of detection for mefloquine in plasma was 10 ng/ml. The mean overall recovery from plasma was 102.7 +/- 3.3%. The method was shown to be specific for mefloquine without any interference from endogenous compounds in plasma or from the drugs pyrimethamine and sulfadoxine (compounds often administered in combination with mefloquine). The assay described was successfully applied to the determination of plasma levels of mefloquine in man and dog following oral and intravenous administration, respectively.     

Quantitative analysis of memantine in human plasma by gas chromatography/negative ion chemical ionization/mass spectrometry

A sensitive and specific method for the determination of memantine in human plasma is presented. Memantine was extracted from plasma and derivatized to the pentafluorobenzoyl derivative in a one-step procedure avoiding any sample concentration steps. Amantadine was used as an internal standard. The compounds were measured by gas chromatography/negative ion chemical ionization mass spectrometry without any further processing. Using this detection mode, the fragment ions at m/z 353 and 325 were obtained at high relative abundance. Calibration graphs were linear over the range 0.117-30 ng ml(-1). At the limit of quantification (LOQ), the inter-assay precision was 2.00% and the intra-assay variability was 3.22%. The accuracy at the LOQ showed deviations of -1.42% (intra-assay) and -2.47% (inter-assay). The method is rugged, rapid and robust and was applied to the batch determination of memantine during pharmacokinetic profiling of the drug.     

Determination of trimecaine metabolites in blood plasma by capillary isotachophoresis

A method is proposed for the determination of trimecaine (diethylglycylmesidide) and its de-ethylated metabolites (monoethylglycylmesidide and glycylmesidide) in blood plasma by capillary isotachophoresis. The deproteinated plasma is extracted into chloroform after alkalinization and the total solids in the organic layer are dissolved in acidified 25% 2-propanol. Subsequent isotachophoretic analysis is performed in an operational system consisting of potassium acetate buffer (pH 4.75) as the leading and beta-alanine as the terminating electrolyte. The order of the zones corresponds to the molecular weights of the separated compounds. The recovery of all substances of interest is 55% and the limit of determination is 0.05 micrograms of each substance in 1 ml of plasma.     

The determination of arsenic and selenium by HPLC combined with alternating current plasma detection and post-column hydride formation

A helium alternating current plasma detector in combination with HPLC has been evaluated for the determination of arsenic and selenium-containing compounds after post-column hydride generation. The construction, operation, and optimization of the system is presented. Detection limits for the compounds under study ranged from 45–60 pg/s. Determination of arsenic and selenium in spiked river water samples has been used to demonstrate the applicability of the technique.     

Quantitative determination of acemetacin and its metabolite indometacin in blood and plasma by column liquid chromatography

A column liquid chromatographic (LC) method using UV detection for the determination of acemetacin and its metabolite indometacin in blood is described. The lower detection limit for both compounds is ca. 25 micrograms/l, the precision (coefficient of variation) is 6% for acemetacin and 10% for indometacin. The method is also suited for determination of both compounds in plasma, precisions in this case are even better than for blood, i.e. around 3% for both acemetacin and indometacin. Blood samples of three volunteers who had received 90 mg of acemetacin orally were analysed using the new method and very good agreement with results from a thin-layer chromatographic/fluorescence method was found.     

Simple and rapid method for the simultaneous determination of the eight main metabolites and conjugates of sulphasalazine in human plasma, urine and faeces using dynamically modified silica

A simple and rapid method for the simultaneous determination of sulphapyridine, N-acetylsulphapyridine, 5-aminosalicylic acid, N-acetyl-5-aminosalicylic acid, hydroxysulphapyridine, N-acetylhydroxysulphapyridine, sulphapyridine O-glucuronide and N-acetylsulphapyridine O-glucuronide in plasma, urine and faeces is presented. After precipitation of plasma proteins by addition of methanol the samples are injected directly into the liquid chromatographic system. The limit of detection is 1 microgram/ml or less at a signal-to-noise ratio of 5 for all compounds using ultraviolet, fluorescence or electrochemical detection. The advantages of the dynamically modified silica approach in this reversed-phase high-performance liquid chromatographic method are demonstrated with respect to regulation and reproducibility of the selectivity.     

Straight-Phase Ion-Pair Chromatography of Zimelidine and Similar Divalent Amines Part I Bioanalysis

Abstract

Methods are presented for the quantitative determination of ZIMELIDINE, a new antidepressant drug, and its active metabolite norZIMELIDINE in biological material (whole blood, plasma, urine and rat brain). The extraction is optimized regarding recoveries and blank chromatograms and the compounds are separated by high performance ion-pair liquid chromagraphy with perchlorate as counter ion in the stationary phase. Internal standards are chlorpheniramine and the geometrical isomer to norzimelidine. The precision for determinations in plasma ranges 2 - 7% (CV) for the concentrations 100 - 5 ng/ml, and the detection limits are 150 pg/ml but can be lowered about five times by using larger sample volumes. The selectivity against metabolites is investigated and the use of the method in routine is discussed. The isolation and identification of the primary amine metabolite by collecting the peak for subsequent GC-MS-analysis is demonstrated.     

High-performance liquid chromatographic determination of morphine and its 3- and 6-glucuronide metabolites: improvements to the method and application to stability studies

Improvements to previously reported methods for the determination of morphine, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) in human plasma are described. The improved methods involve the use of a solid-phase extraction cartridge and a chromatographic system which uses paired-ion reversed-phase high-performance liquid chromatography with a radially compressed column. Only one cartridge is used to prepare each sample for chromatography and each cartridge may be used for at least fourteen 1-ml plasma samples. The recovery is greater than 85%. The improvements to the method of sample pretreatment and in the chromatographic conditions have allowed determination of morphine, M3G and M6G in human plasma down to 13.3 nmol/l (coefficient of variation = 9.3%), 108 nmol/l (6.6%) and 41 nmol/l (6.7%), respectively, using ultraviolet detection alone. It was shown that all three compounds were stable in plasma for up to 101 weeks when stored at -20 degrees C.     

High-performance liquid chromatographic determination of L-3-(3,4-dihydroxyphenyl)-2-methylalanine (alpha-methyldopa) in human urine and plasma

A procedure is described for the determination of alpha-methyldopa (MD) [L-3-(3,4-dihydroxyphenyl)-2-methylalanine], its metabolite and catecholamines in the urine and plasma of patients undergoing MD therapy, by high-performance liquid chromatography with dual working electrode coulometric detection. An efficient sample preparation procedure is presented for the isolation of endogenous MD, its metabolite and catecholamines from plasma or urine. After deproteinization of a plasma sample with methanol containing 2% of 0.5 M perchloric acid and dilution of a urine sample (1:200), MD, dihydroxyphenylacetic acid (DOPAC), 3-O-methylmethyldopa (3-OMMD) and homovanillic acid (HVA) were separated with a Supelcosil LC-18 column. Catecholamines were extracted from the supernatant of deproteinized plasma or from urine by ion exchange on a Sephadex CM-25 column and subsequent adsorption on alumina. The use of the same mobile phase for the concurrent assay of MD, its metabolite and catecholamines increased considerably the efficiency of sample separation. Recoveries were close to 100% for MD, DOPAC, 3-OMMD and HVA and 70% for catecholamines. The effects of various experimental parameters related to mobile phase composition on chromatographic performance are reported. The purity of the eluted compounds was tested by recording both the first detector response (oxidation current) and the second detector response (reduction current). The ratio of the detector responses yielded a chemical reversibility ratio for the detected compound. A number of applications such as monitoring data from patients under MD therapy are presented.     

Fast screening method for the determination of angiotensin II receptor antagonists in human plasma by high-performance liquid chromatography with fluorimetric detection

A selective, accurate and precise high-performance liquid chromatographic assay coupled to fluorescence detection was developed for the detection of some angiotensin II receptor antagonists (ARA II): Losartan, Irbesartan, Valsartan, Candesartan cilexetil and its metabolite Candesartan MI. The analytes and the internal standard (bumetanide, a high-ceiling diuretic) were extracted from plasma under acidic conditions by means of solid-phase extraction using C8 cartridges. This procedure allowed recoveries close to 80% for all these drugs excluding Candesartan cilexetil (70%) which presented adsorption processes on glass and plastic walls. The analytes and potential interferences were separated on a reversed-phase column, muBondapak C18, at room temperature. A gradient elution mode was used to carry out the separation, the optimal mobile phase being composed of acetonitrile-5 mM acetate buffer, pH 4, at variable flow-rates (from 1.0 to 1.2 ml/min). Fluorescence detector was set at an excitation wavelength of 250 nm and an emission wavelength of 375 nm. Intra- and inter-day relative standard deviations for all the compounds were lower than 8% except for Losartan (12%) and the method assesses a quite good accuracy (percentage of relative error approximately 6% in most of the cases). The limit of quantitation for these compounds was 3 ng/ml for Candesartan cilexetil and M1, 16 ng/ml for Losartan and 50 ng/ml for Irbesartan and Valsartan, which allows their determination at expected plasma concentration levels. This assay method has been successfully applied to plasma samples obtained from hypertensive patients under clinical studies after oral administration of a therapeutic dose of some of these ARA II compounds.     

Determination of carnitine and acylcarnitines in plasma by high-performance liquid chromatography/electrospray ionization ion trap tandem mass spectrometry

A high-performance liquid chromatography/mass spectrometry method was developed for the determination of carnitine, its biosynthetic precursor butyrobetaine, and eight acylcarnitines in plasma. The procedure includes a solid-phase extraction for carnitine and short- and medium-chain acylcarnitines, and a liquid-liquid extraction for protein-bound long-chain acylcarnitines, followed by separation on a reversed-phase column in the presence of a volatile ion-pairing reagent. Detection was achieved using an ion-trap mass spectrometer run in the tandem mass spectrometry (MS/MS) mode. The choice of the matrix for calibrators, used for quantification of these endogenous compounds, was also investigated. Validation was performed for standard quality controls diluted with 4% bovine serum albumin solution and for spiked plasma quality control samples at concentrations between 0.5 and 80 micromol/L, depending on the compound. Intra- and inter-day precisions for the determination of carnitine were below 3.4% and accuracies were between 95.2 and 109.0%. Application of the method to the diagnosis of pathological acylcarnitine profiles of metabolic disorders in a patient suffering from methylmalonic aciduria is presented. The method allows quantification of carnitine, butyrobetaine, acetylcarnitine and propionylcarnitine, and semiquantitative analysis of medium- and long-chain acylcarnitines. In contrast with other methods, no derivatization step is needed.     

A rapid and precise microheterometric determination of mercury with p-dimethylamino-benzylidene-rhodanine

1. In citrate or tartrate solutions mercury formed three different compounds with p-dimethyl-amino-benzylidene-rhodanine at pH ~5,~7 and ~9.The composition of the compounds is discussed. 2. Analytical methods are presented for the determination of 0.1 mg mercury in 20 ml solution which contained 99.5–99.9% foreign metals. In most cases the error lay between zero and 1%. The foreign metals were :Ca, Ba, Mg, Zn, Mn, Ni, Co, Al, Cr(III), Fe(III), Cd, Cu, Pb, U(VI), Tl(I), Th, Ce(III), Sb(III) and Bi(III). Methods for the determination of 0.1 mg mercury in gold (III), Pt(VI), Pd(II) and silver are also presented.     

Gas Liquid Chromatographic Determination of Acetaminophen in Blood Plasma

Abstract

An improved procedure for the GLC determination of acetaminophen (N-acetyl-p-aminophenol) in the presence of N-butyryl-p-aminophenol (internal standard) is described. The method is based on the extraction of acetaminophen from plasma with ethyl acetate containing a known amount of N-butyryl-p-aminophenol. Following a clean-up step with a basic buffer solution and neutralization with acid, both compounds are reextracted into ethyl acetate. The ethyl acetate is evaporated to dryness and the residue dissolved in 5 μl of pyridine and 15 μl of acetic anhydride at 42°C. One to 2 μl samples are injected directly into the gas chromatograph. This extraction process does not give rise to troublesome interfering peaks in the chromatogram. In addition, it prevents late-eluting peaks which inhibit efficient processing of samples. The recovery of acetaminophen is approximately 54%, and the limit of quantitation 0.5 μg/ml of plasma. Data are presented to illustrate the practicality of the method for bioavailability evaluation from acetaminophen plasma levels after oral administration of 325 mg of an acetaminophen dosage form.     

High performance liquid chromatography with UV detection for the simultaneous determination of sympathomimetic amines using 4-(4,5-diphenyl-1H-imidazole-2-yl)benzoyl chloride as a label

A high performance liquid chromatographic method has been developed for the simultaneous determination of (+/-) fenfluramine (Fen) and phentermine (Phen) in addition to three other sympathomimetic amines-ephedrine (E), norephedrine (NE) and 2-phenylethylamine (2-PEA), using cyclohexylamine (CX) as an internal standard in plasma. The compounds were derivatized with 4-(4,5-diphenyl-1H-imidazole-2-yl)benzoyl chloride (DIB-Cl) to give the DIB-derivatives. The derivatives were then separated using an isocratic HPLC system with UV detection. The limits of detection for Fen, Phen, E, NE and 2-PEA in plasma ranged from 0.32 to 22.9 pmol on column at a signal-to-noise ratio of 3. The recoveries following alkaline extraction from plasma samples of known concentrations were found to be more than 94% for the studied compounds. This method might be useful for the screening of the studied sympathomimetic amines in human plasma samples in forensic as well as toxicological studies. Furthermore, the developed method was modified for the simultaneous determination of Fen and Phen in human and rat plasma using fluoxetine as an internal standard. The methods are reproducible and precise. Finally, the two drugs were administered intraperitoneally to rats in combination, and their plasma levels over the investigated time course were successfully determined.     

Simultaneous determination of sulfamethoxazole and trimethoprim in human plasma by capillary zone electrophoresis

A capillary electrophoretic method for the simultaneous determination of sulfamethoxazole and trimethoprim in plasma was developed. Sulfamethoxazole and trimethoprim extracted from human plasma with ethyl acetate were analyzed at 20 kV and 25 degrees C using 15 mm phosphate buffer (pH 6.2) as the electrolyte. The detection was by UV at 220 nm. The run time was 8.0 min and the limit of quantification was 10.00 microg/mL for sulfamethoxazole and 2.00 microg/mL for trimethoprim. The recovery was >99% for both compounds. This method enabled the detection of sulfamethoxazole and trimethoprim in plasma of patients after oral ingestion of their combined formulation. The present simple and rapid method is applicable to drug monitoring in immunocompromised patients who are taking the combined formulation of these compounds for the treatment or prophylaxis of Pneumocystis carinii pneumonia.     

Determination of neodymium and boron in iron-neodymium-boron alloys by direct-current plasma atomic-emission spectrometry

Rapid, accurate methods for the determination of neodymium and boron in iron-neodymium-boron alloys are presented. The procedure involves dissolution of the alloy with nitric acid and measurement of the elemental concentrations by direct-current plasma atomic-emission spectrometry. Lithium is added in the determination of neodymium, to control interferences. The relative standard deviations are approximately 1%, and the accuracies comparable to those obtained by classical chemical methods.     

An HPLC Method for the Determination of Diltiazem and Desacetyldiltiazem in Human Plasma

Abstract

A high performance liquid chromatographic method is presented for the determination of diltiazem and its metabolite desacetyldiltiazem in human plasma. Diltiazem and desacetyldiltiazem are extracted from plasma basified with 0.5M dibasic sodium phosphate (pH 7.4) using 1% 2-propanol in n-hexane containing diazepam as an internal standard. A reversed phase cyanopropylsilane column was used with a mobile phase of 45% acetonitrile and 55% 0.05M acetate buffer (pH 4.0). The minimum detectable limit was 2ng/ml of plasma. The effect of the pH, molarity, and percent acetonitrile of the mobile phase on the capacity factor was studied. Possible interferences from other drugs administered concurrently are presented.