Simultaneous determination of oleuropein and its metabolites in plasma by high-performance liquid chromatography

A method based on high-performance liquid chromatography (HPLC) with a diode array detection system was developed and validated aiming at the simultaneous determination of oleuropein (OE) and its metabolites, hydroxytyrosol (HT) and tyrosol (T), in human plasma. These phenolic components are believed to play a vital role in the prevention of coronary artery disease and atherosclerosis. The proposed method includes a clean-up solid-phase extraction procedure (using a C(18) column) with high recovery efficiency (85-100%). The statistical evaluation of the method reveals good linearity, accuracy and reproducibility for all the compounds analyzed with RSD values less than 6.5%, while the detection limit is 50 ng/ml for both OE and T and 75 ng/ml for HT. This assay can be employed in bioavailability studies of olive oil phenolic compounds, thus assisting the evaluation of their pharmacological role.     

Use of metal chelate affinity chromatography and membrane-based ion-exchange as clean-up procedure for trace residue analysis of tetracyclines in animal tissues and egg

A new and efficient procedure for the clean-up of tetracycline residues in animal tissues and egg prior to reversed-phase high-performance liquid chromatography is described. The principal steps involve homogenization of the tissues in sodium succinate buffer and methanol, followed by centrifugation and clean-up with metal chelate affinity chromatography (MCAC). After further concentration on an Empore extraction membrane with cation-exchange properties, the sample is analysed by HPLC with fluorescence detection. The method was tested on porcine kidney and muscle, bovine liver and whole chicken's egg. The recoveries were determined from spiked tissues for oxytetracycline, tetracycline, chlortetracycline and doxycycline and ranged from 40 to 70%, with repeatabilities below 10% R.S.D.. The analytical responses were linear in the range up to at least 1000 ng/g. The detection limits of the method were estimated at 0.42 ng/g of oxytetracycline, 0.49 ng/g of tetracycline, 0.66 ng/g of chlortetracycline and 1.38 ng/g of doxycycline in porcine muscle, using signal-to-noise ratios of 4:1. Similar detection limits were estimated for kidney, liver and egg. The measured limits of quantification were 2 ng/g for oxytetracycline, 3 ng/g for tetracycline, 4 ng/g for chlortetracycline and 5 ng/g for doxycycline in porcine kidney. The advantage of this method over existing methods is its increased limit of detection.     

Sensitive assay for the tricyclic antidepressant Ro 11-2465 in biological fluids by high-performance liquid chromatography and fluorescence detection

A high-performance liquid chromatographic assay, suitable for pharmacokinetic studies, has been developed for the new tricyclic antidepressant Ro 11-2465, at present under clinical investigation. For concentrations above 0.5 ng/ml, the method involves a simple extraction at basic pH with an organic solvent followed by direct chromatography of this extract on a silica gel column using fluorescence detection. For concentrations below 0.5 ng/ml, an extensive clean-up procedure is required. In both procedures, however, evaporation of the extract and reconstitution of the residue is avoided. The detection limit, using 1 ml of plasma, is about 0.1 ng/ml. This sensitivity is sufficient for following single-dose kinetics of Ro 11-2465 in man.     

Automated determination of clomipramine and its major metabolites in human and rat serum by high-performance liquid chromatography with on-line column-switching

A fully automated method including column-switching and isocratic high-performance liquid chromatography (HPLC) was developed for simultaneous determination of the tricyclic antidepressant clomipramine and its metabolites demethylclomipramine, 2-, 8-, and 10-hydroxyclomipramine, 2-, and 8-hydroxydemethylclomipramine and didemethylclomipramine in serum. After serum injection into the HPLC system and on-line sample clean-up on a clean-up column (Hypersil CN; 10×4.6 mm) by an eluent consisting of 35% acetonitrile and 65% deionized water, the chromatographic separation was performed on an analytical column (LiChrospher CN; 250×4.6 mm I.D.) by an eluent consisting of 38% acetonitrile and 62% aqueous sodium perchlorate (0.02 M, pH 2.5). The UV detector was set at 260 nm. The limit of quantification was about 15 ng/ml for all analytes. The coefficients of variation ranged between 3 and 12% with recovery rates between 64 and 110%. Linear regression analyses revealed coefficients of correlation between 0.98 and 0.99. The method could be applied to therapeutic drug monitoring as well as metabolism studies in man and rat.     

Determination of hydroxy metabolites of polychlorinated biphenyls in plasma and tissue by gas chromatography/mass spectrometry

This study examines a novel sample preparation method for the determination of 11 hydroxy metabolites of polychlorinated biphenyls (PCBs) in plasma and organ tissues, followed by gas chromatography with mass spectrometric detection (GC/MS). The clean-up method was optimized to eliminate the interference matter by using a silica column and 10 mL of n-hexane/dichloromethane (4:6, v/v) as an eluent. Solid-phase and solvent extraction procedures were used for the plasma and tissues samples, respectively. Compared to C(18) and C(8) solid-phase, C(2) showed higher extraction efficiency with n-hexane as the eluent for plasma. The hydroxy-PCB extraction recoveries achieved with this combined extraction and clean-up procedure from plasma ranged from 87 to 117%, while those from tissues ranged from 82 to 111%. The linear detector responses for propyl derivatives of hydroxy-PCBs were obtained with the coefficients of determination varying from 0.992 to 0.998 in the concentration range of 0.1-20 ng mL(-1). The method detection limits ranged from 0.1 to 0.5 ng mL(-1) in 1 mL of plasma and from 0.1 to 0.5 ng g(-1) in 1g of tissues. This procedure was successfully applied to the study of 3-OH-2,3',4,4',5-PeCB in rat plasma and liver samples after intraperitoneal injection (20 mg/kg) of 2,3',4,4',5-PeCB.     

High throughput method for the determination of organochlorine pesticides and polychlorinated biphenyls in human serum

An improved method for the determination of selected organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) in human serum was developed. The method requires low volume of serum (500 microl) and 48-96 samples per day can be prepared by one analyst without special automatic equipment. Initial extraction was performed using 96-well solid-phase extraction disk plates and was followed by a clean-up with silica gel/sulfuric acid. Different denaturation, elution and clean-up conditions were tested. Quantification was carried out by gas chromatography equipped with electron capture detector (GC-ECD) or mass spectrometer (GC-MS). Recoveries of PCB congeners 28, 52, 101, 118, 138, 153 and 180 and OCPs HCB, beta-HCH, p,p'-DDE and p,p'-DDT at two spiking levels (n=8) varied from 57 to 120%, and intra-day relative standard deviation from 1 to 11%, both depending on spiking level and compound. Inter-day relative standard deviation was <15% in all cases. Limit of quantification (LOQ) for these PCBs ranged from 0.08 to 0.13 ng/ml and for these OCPs from 0.16 to 0.40 ng/ml. The optimized method was applied to the analysis of 1000 serum samples from different places of Spain.     

Development of an analytical approach for identification and quantification of 5-α-androst-16-en-3-one in human milk

A method was developed for the quantification of 5-α-androst-16-en-3-one in human breast milk based on application of a stable isotope dilution assay using 5α-androst-16-en-3-one-6, 6-d₂. The procedure includes extraction of the human milk by hexane with subsequent clean-up of the obtained extract by gel permeation and silica gel column chromatography. The extracted samples were analyzed by gas chromatography–mass spectrometry. Using this method 5-α-androst-16-en-3-one could be identified and for the first time quantified in a concentration range of 26–155 ng/kg in human milk.     

Determination of aloesin in rat plasma using a column-switching high-performance liquid chromatographic assay

A column-switching high-performance liquid chromatography (HPLC) method for the determination of aloesin in rat plasma using column-switching and ultraviolet (UV) absorbance detection is described. Plasma was directly injected onto the HPLC system consisting of a clean-up column, a concentrating column, and an analytical column, which were connected with a six-port switching valve. The determination of aloesin was accurate and repeatable, with a limit of quantitation of 10 ng/ml in plasma. The standard calibration curve for aloesin was linear (r=0.998) over the concentration range of 10–1000 ng/ml in rat plasma. The intra- and inter-day assay variabilities of aloesin ranged from 1.0 to 4.7% and 1.1 to 8.8%, respectively. This highly sensitive and simple method has been successfully applied to a pharmacokinetic study after oral administration of aloesin to rats.     

Determination of beta-sympathomimetics in liver and urine by immunoaffinity chromatography and gas chromatography—mass-selective detection

A specific and sensitive method for the determination of several β-agonistic drugs in liver and urine is described. Following clean-up by immunoaffinity chromatography and two different derivatizations, gas chromatography—mass spectrometry with electron-impact ionization is performed. The immunoaffinity chromatography columns were packed with Sepharose-immobilized polyclonal antibodies raised against the β-agonist clenbuterol. Owing to the high clean-up efficiency of the immunoaffinity column large sample volumes can be used (up to 100 ml urine or 25 gram liver). The immunoaffinity sample pretreatment is highly specific and no further sample pretreatment was necessary. Due to the combination of two different derivatizations only GC—MS with electron-impact ionization is necessary to fulfil legal requirements. The first confirmation step consists of a derivatization reaction between the hydroxyl group of the parent compound and trimethylsilane. The second confirmation method is a derivatization to a cyclic derivative with the hydroxyl group and the aliphatic nitrogen group. Limits of determination in liver as well in urine are at the 10 ng/kg or ng/l (ppt) level with acceptable signal-to-noise ratio. The method is suitable for identification and quantification of trace amounts of several similar β-agonistic drugs either used separately or in combination and can be used also for quantification of clenbuterol in liver with regard to levels exceeding the maximum residue limit (MRL) of 1 μg/kg (ppb).     

Determination of ivermectin in bovine liver by optical immunobiosensor

A rapid and sensitive biosensor immunoassay was developed for residues of the antiparasitic agent ivermectin in bovine liver. A detection limit of 19.1 ng g(-1) was achieved. The sensor employed was a Biacore optical instrument based on surface plasmon resonance. 5-O-succinoylivermectin-apo-transferrin conjugate was used to produce monoclonal antibody while a second derivative, ivermectin-oxime, was immobilised onto the surface of a sensor chip. A range of assay parameters (flow rate, injection time, temperature) and extraction techniques were investigated. In the final assay procedure, ivermectin was extracted with acetonitrile followed by C(8) SPE clean-up. Matrix effect was minimised by increasing the flow rate to 25 microl min(-1) and reducing the sample injection time to 2 min. The average value for liver samples spiked at 100 ng g(-1) (the MRL for the drug) and 50 ng g(-1) concentrations were 93.7 and 43.2 ng g(-1), respectively.     

Determination of Ochratoxin A in small volumes of human blood serum

A new simple and rapid method for analysing Ochratoxin A (OTA) in small volumes of human blood serum using capillary zone electrophoresis coupled to laser-induced fluorescence is described. The clean-up procedure solely consists of a double extraction step. To improve the reproducibility of migration times and quantification, two internal standards were used. The limit of detection was 0.55 ng/ml, with a linear range of 1-100 ng/ml of OTA in spiked human blood serum. The method is used to rapidly screen suspected patients.     

Direct determination of estriol 3- and 16-glucuronides in pregnancy urine by column-switching high-performance liquid chromatography with fluorescence detection

An HPLC method for the direct and simultaneous determination of estriol 3- and 16-glucuronides in pregnancy urine is described. The method is based on direct derivatization of the glucuronic acid moiety in estriol glucuronides in urine with 6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone-3-propionylcarboxylic acid hydrazide. The derivatization reaction proceeds in aqueous solution (or urine sample) in the presence of pyridine and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide at 37°C. The resulting fluorescent derivatives were separated by column-switching chromatography using a first column (YMC-Pack C₄) for clean-up of the derivatives and a second column (YMC Pack Ph) for the complete separation of the derivatives. The derivatives were detected spectrofluorimetrically at 445 nm with excitation at 367 nm. The detection limits (signal-to-noise ratio=3) for estriol 3- and 16-glucuronides were 150 and 180 fmol in a 5 μl of urine (14 and 17 ng ml⁻¹ urine), respectively. The present method is highly sensitive and simple without any clean-up such as conventional solid-phase extraction.     

Quantitation of 6-mercaptopurine in biologic fluids using high-performance liquid chromatography: A selective and novel procedure

A simple, selective, sensitive and rapid procedure is described for the quantitation of 6-mercaptopurine (6-MP) in biological fluids. A sensitivity of at least 5 ng/ml is easily achieved in plasma on a reversed-phase octadecylsilane (C₁) column using a high-performance liquid chromatography system following an initial protein precipitation and a clean-up step. Mean extractability of the drug from plasma following this procedure is greater than 98% and the overall coefficient of variation for the assay is below 6%. Plasma levels of 6-MP were measured in a rhesus monkey for 12 h following an intravenous administration of a single bolus dose (4 mg/kg) of 6-MP.     

Development and validation of a sensitive HPLC-ESI-MS/MS method for the direct determination of glucosamine in human plasma

A sensitive and specific HPLC-ESI-MS/MS method for the direct determination of glucosamine in human plasma has been developed and validated. Plasma samples were analyzed after a simple, one-step protein precipitation clean-up with trichloroacetic acid using a polymer-based amino high-performance liquid chromatography (HPLC) column and a water/acetonitrile mobile phase elution gradient, with d-[1-(13)C]glucosamine as the internal standard. Detection was performed by mass spectrometry, using an electrospray source and employing multiple reaction monitoring to separately monitor glucosamine and the internal standard. The limit of quantification of the method was 10ng/ml of glucosamine and the calibration curve showed a good linearity up to 1000ng/ml. The precision (R.S.D.) and the accuracy (bias) of the method at the limit of quantification were 13.8 and 4.0%, respectively, and the mean recovery of glucosamine at three concentration levels was 101.6+/-5.7%. The method was applied for the determination of glucosamine concentrations in human plasma samples collected from untreated healthy volunteers and, in a separate bioavailability study, to evaluate plasma glucosamine pharmacokinetics profiles after oral administration of crystalline glucosamine sulfate.     

Determination of organochlorine pesticides and polychlorinated biphenyls in human serum using headspace solid-phase microextraction and gas chromatography-electron capture detection

A simple procedure for the determination of organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) in human serum using headspace solid-phase microextraction (HS-SPME) was developed. The analysis was carried out by gas chromatography (GC) equipped with electron capture detector (ECD). A 2(7-4) Plackett-Burman reduced factorial design for screening and a central composite design for optimizing the significant variables were applied. A 100 microm PDMS fiber, 3/5 headspace ratio (3 ml in 5 ml vial), 85 degrees C extraction temperature, 50 min extraction time, and 1 ml of acidic solution (pH 3) added to 1 ml of diluted serum (1:1) were chosen for the best response in HS extraction mode. The detection limits found were from 1 pg/ml (PCB 167) to 52 pg/ml (beta-HCH), the relative standard deviation for the procedure varied from 3% (PCB 52) to 12% (PCB 189) and the accuracy was checked by using validated solid-phase extraction (SPE) procedure. The method that avoids the use of clean-up steps and the hazardous solvents enabled reliable determinations of the OCPs and the PCBs except beta-HCH. The method was applied to the analysis of 33 human serum samples. The most abundant target compound was p-p'-DDE (range, 0.3-8.0 ng/ml; median value, 2.1 ng/ml). Among the PCBs the prevalent congeners were 138, 153 and 180.     

Automated column-switching high-performance liquid chromatography for the determination of aflatoxin M1

An extractionless method for determining aflatoxin M1 (AFM1), a major metabolite of aflatoxin B1 (AFB1), in human urine was developed. The biological fluid is injected directly into the chromatographic system after simple dilution and centrifugation. A pre-column, packed with a cation-exchange phase and coupled on-line to a column-switching liquid chromatography (LC) system, is used for sample pre-treatment and concentration. The analytes are non-selectively desorbed with the LC eluent and cleaned by means of a column-switching procedure. Pre-treatment and analysis were performed within 40 min. Average AFM1 recovery reached 97% in the 10–100 ng/l range of urine. The detection limit of AFM1 in urine and milk was 2.5 ng/l for 1 ml of injected sample. A comparison with an immunoaffinity column clean-up and LC method was performed. The method was applied to determine AFM1 in the urine of AFB1 gavaged rats, and in the urine of both potentially exposed and supposedly unexposed workers. The method was also extended to milk.     

Sensitive gas chromatographic method for determination of mercapturic acids in human urine

A method was developed for sensitive determination of the specific benzene metabolite S-phenylmercapturic acid and the corresponding toluene metabolite S-benzylmercapturic acid in human urine for non-occupational and occupational exposure. The sample preparation procedure consists of liquid extraction of urine samples followed by precolumn derivatization and a clean-up by normal-phase HPLC. Determination of analytes occurs by gas chromatography with electron-capture detection. With this highly sensitive method (detection limits 60 and 65 ng/l, respectively) urinary S-phenylmercapturic and S-benzylmercapturic acid concentrations for non-occupationally exposed persons (e.g. non-smokers) can be measured precisely in one chromatographic run. Validation of the method occured by comparison with a HPLC method we have published recently.     

Simultaneous determination of residual stilbenes and stilbene metabolites in animal tissue by liquid chromatography-tandem mass spectrometry

A liquid chromatographic (LC)-tandem mass spectrometry (MS/MS) method was developed for simultaneous determination of stilbenes, diethylstilbestrol (DES), hexestrol (HEX), and dienoestrol (DEN) in animal tissue. Sample clean-up and analyte enrichment was performed by automated solid-phase extraction (ASPE) with a silica gel cartridge. Detection capabilities (CCbeta) related to the transition products of lowest abundance for the method were 0.04-0.45 ng g(-1) in tissue and were achieved using atmospheric pressure chemical ionization (APCI) in negative mode. The use of an internal standard in combination with the simplified sample preparation led to a sensitive and reliable analytical method. The recovery level of the method was 84-108% for DES and DEN between 0.5 and 5 ng g(-1), and 59-87% for HEX between 0.25 and 2.5 ng g(-1).     

Molecularly imprinted polymer cartridges coupled on-line with high performance liquid chromatography for simple and rapid analysis of dextromethorphan in human plasma samples

In this paper, a novel method is described for automated determination of dextromethorphan in biological fluids using molecularly imprinted solid-phase extraction (MISPE) as a sample clean-up technique combined with high performance liquid chromatography (HPLC). The water-compatible molecularly imprinted polymers (MIPs) were prepared using methacrylic acid as functional monomer, ethylene glycol dimethacrylate as cross-linker, chloroform as porogen and dextromethorphan as template molecule. These imprinted polymers were used as solid-phase extraction sorbent for the extraction of dextromethorphan from human plasma samples. Various parameters affecting the extraction efficiency of the MIP cartridges were evaluated. The high selectivity of the sorbent coupled to the high performance liquid chromatographic system permitted a simple and rapid analysis of this drug in plasma samples with limits of detection (LOD) and quantification (LOQ) of 0.12 ng/mL and 0.35 ng/mL, respectively. The MIP selectivity was evaluated by analyzing of the dextromethorphan in presence of several substances with similar molecular structures and properties. Results from the HPLC analyses showed that the recoveries of dextromethorphan using MIP cartridges from human plasma samples in the range of 1-50 ng/mL were higher than 87%.