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PubMed2005

Determination of colchicine residues in sheep serum and milk using high-performance liquid chromatography combined with electrospray ionization ion trap tandem mass spectrometry.

Colchicine is a naturally occurring alkaloid used in human and veterinary medicine. It shows genotoxicity in in vitro and in vivo systems even at low concentrations. Therefore, no ADI has been established, and colchicine has been included in Annex IV of Council Regulation (EEC) No. 2377/90. No abuse of this drug in intensive livestock farming has yet been reported. However, there may be a natural route of entry for this compound into the food chain when Colchicum autumnale is consumed by animals kept outdoors. To address this concern, we developed and validated a highly sensitive and selective quantitative LC-ESI-MS-MS method for the detection of colchicine in sheep serum and milk. For sample pretreatment, all samples were liquid-liquid extracted with phosphate buffer (pH 8.0) and dichloromethane. LC separation was carried out on an RP C18 column employing a 0.5% formic acid/acetonitrile gradient system. The recoveries in both matrixes at a concentration range from 0.0005 to 1 mg/L were >80% with RSDs of <10%. The applicability of the method was demonstrated in serum and the corresponding milk samples from Albanian sheep. After exposure to C. autumnale, the animals showed clinical signs of intoxication. Significant concentrations (up to 1 mg/L) of the drug were found in serum as well as in milk samples.

Animals↗

PubMedDate not supplied

Microparticle-enhanced nephelometric immunoassay with microsphere-antigen conjugates.

gamma-Irradiation of acrolein and other acrylic monomers allowed the synthesis of spherical polyfunctional hydrophilic microparticles in the size range of 50 to 300 nm, on which antigens (immunoglobulins G, chorionic gonadotropin hormone, prealbumin) could be covalently bound. Microsphere-antigen conjugates clustered together in the presence of specific antiserum or monoclonal antibodies and their agglutination was quantified by light-scattering measurement performed with a specially designed nephelometer. Essential factors concerning the conjugate agglutination and its quantitation (size of microsphere, amount of antigen bound on microsphere, concentration of conjugate, concentration of agglutinating reagent, angle of light-scattering observation) were successively studied. A microparticle-enhanced nephelometric immunoassay for prealbumin was finally developed as an example of application. It was based on the inhibition of the immunoagglutination of microspheres-prealbumin conjugate by free prealbumin. This prealbumin immunoassay was easy to perform (one-step assay without washing or phase separation), fast (30 min), reliable (variation coefficients ranged from 3.6% to 7.5% for within- and between-assay determination), and sensitive (1 microgram/L detected). It was correlated with conventional immunonephelometry and radial immunodiffusion (correlation coefficients, 0.98). Microparticle-enhanced nephelometric immunoassay offered many advantages over the last two methods. Its better sensitivity allowed a lower reagent consumption and a larger sample dilution (contrary to the conventional immunonephelometry, sample pretreatment and sample blank measurement were unnecessary). Its inhibition mode induced a total accuracy for sample with high analyte concentration (a risk of underevaluation in antigen excess conditions existed in all method based on a noncompetitive antigen-antibody reaction) and provided the possibility to quantify haptens.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrolein↗

PubMed2006

Quantitative analysis of tylosin in eggs by high performance liquid chromatography with electrospray ionization tandem mass spectrometry: residue depletion kinetics after administration via feed and drinking water in laying hens.

UNLABELLED: Maximum residue limits (MRLs) have been established by the European Union when tylosin is used therapeutically. They are fixed at 200 microg/kg for eggs. A highly sensitive and selective quantitative liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) method suitable for monitoring tylosin residues in eggs to determine its depletion kinetics was developed and validated. For sample pretreatment all samples were liquid-liquid extracted with citrate buffer (pH 5.0) and acetonitrile. Liquid chromatographic separation was carried out on a reversed phase C18 column employing a 0.5% formic acid/acetonitrile gradient system. The tylosin recovery in eggs at a concentration range from 1.0-400 microg/kg was >82% with relative standard deviations between 1.5 and 11.0%. In two experimental studies administrating tylosin via feed (final dosage: 1.5 g/kg) or drinking water (final dosage: 0.5 g/L), no residues above the MRL were found during and after treatment. Moreover, all samples were well below the actual MRL of 200 microg/kg. Therefore, our residue data suggest that a withholding period for eggs is not required when laying hens are treated with tylosin in recommended dosages via feed or drinking water. KEYWORDS: Tylosin; residue; depletion; laying hen; withholding period; mass spectrometry.

Animal Feed↗

PubMed1996

Concomitant mobilization of plasma cells and hematopoietic progenitors into peripheral blood of patients with multiple myeloma.

In this article, we review neoplastic contamination in the peripheral blood (PB) of patients with multiple myeloma (MM) upon stem cell mobilization. We first evaluated PB samples from pretreated MM patients following administration of high-dose cyclophosphamide (Cy, 7 g/m2 or 4 g/m2) and granulocyte colony-stimulating factor (G-CSF) for the presence of myeloma cells as well as hematopoietic progenitors. Plasma cells containing intracytoplasmic immunoglobulin (cIg) were counted by immunofluorescence microscopy after incubation with appropriate antisera against light and heavy chain Ig. Flow cytometry studies were performed to determine the presence of malignant B lineage elements, using monoclonal antibodies against the CD19 antigen and the monotypic light chain. Prior to PBSC mobilization, circulating plasma cells were detected in all MM patients at 0.1%-1.8% of the mononuclear cell (MNC) fraction (mean value 0.7 +/- 0.4% SD). In these patients, a higher absolute number of PB neoplastic cells was detected after administration of chemotherapy and G-CSF. Kinetic analysis showed a pattern of tumor cell mobilization similar to that of normal hematopoietic progenitors, with the peak coinciding with the optimal period for the collection of PBSC. The absolute number of plasma cells showed a 10-50-fold increase over the baseline value. Apheresis products contained 0.7 +/- 0.2% SD myeloma cells (range 0.2%-2.7%), which demonstrated the capacity of plasma cells to proliferate, differentiate, and mature in response to c-kit ligand (SCF), IL-3, IL-6, and a combination of IL-3 and IL-6. Subsequently, in an attempt to reduce tumor cell contamination prior to autologous transplantation, circulating hematopoietic CD34+ cells were highly enriched by avidin-biotin immunoabsorption, cryopreserved, and used to reconstitute bone marrow (BM) function after myeloablative therapy in 13 patients. The median purity of the enriched CD34+ cell population was 89.5% (range 51%-94%), with a 75-fold enrichment compared with the pretreatment samples. The median overall recovery of CD34+ cells and CFU-GM was 58% (range 33%-95%) and 45% (range 7%-100%), respectively. Positive selection of CD34+ cells resulted in 2.5-3 log depletion of plasma cells and CD 19+ B lineage cells as determined by immunofluorescence studies, although DNA analysis of the CDR III region of the IgH gene demonstrated the persistence of minimal residual disease (MRD) in 5 of 6 patient samples studied. Myeloma patients were reinfused with enriched CD34+ cells after myeloablative therapy consisting of total body irradiation (TBI, 1000 cGy) and high-dose melphalan (140 mg/m2) or melphalan (200 mg/m2) alone. They received a median of 5 x 10(6) CD34+ cells/kg and showed a rapid reconstitution of hematopoiesis. The median time to 0.5 x 10(9) neutrophils, 20 x 10(9) and 50 x 10(9) platelets/L of PB was 10, 11, and 12 days, respectively. These results, as well as other clinically significant parameters, did not significantly differ from those of patients (n = 13) receiving unmanipulated PBSC following the same pretransplant conditioning regimen. Our data demonstrate the concomitant mobilization of tumor cells and hematopoietic progenitors in the PB of MM patients. Positive selection of CD34+ cells reduces the contamination of myeloma cells from the apheresis products up to 3 log and provides a cell suspension capable of restoring normal hematopoiesis following a TBI-containing conditioning regimen.

Antigens, CD19↗

PubMed1992

Multiple biological markers in germ cell tumor patients treated with platinum-based chemotherapy.

Blood samples from 36 germ cell tumor patients receiving chemotherapy with either cisplatin or carboplatin in combination with other drugs [etoposide or vinblastine, cyclophosphamide, dactinomycin, and bleomycin (VAB-6)] were analyzed for the presence of 7 different biological markers. The biomarkers included platinum-protein adducts, platinum-DNA adducts, sister chromatid exchange (SCE), micronuclei (MN), and somatic gene mutation at the hypoxanthine phosphoribosyl transferase (HPRT) locus and the glycophorin A (GPA) loci (NO and NN). Patients were asked to donate 9 serial blood samples: a pretreatment sample followed by another drawn 12-24 h after each of four cycles of treatment and a final sample provided 3-6 months after the last cycle. Most individuals gave 7-8 samples; 7 individuals donated all 9. Because of limited amounts of cells in some cases, it was not possible to carry out all 7 assays on every sample. Pt-protein adducts, Pt-DNA adducts, and SCE showed a direct and consistent effect of treatment and were very highly correlated. A significant correlation was also seen between both Pt-protein and Pt-DNA adducts and HPRT mutation. All of the posttreatment samples were significantly elevated compared to the baseline sample. These markers also remained elevated 3-6 months after the end of treatment. By contrast, MN, HPRT mutation, and GPA mutation (both NO and NN variants) showed varying patterns of dose response, probably reflective of the differing biology of these markers scored in lymphocytes (MN and HPRT) and erythrocytes (GPA). MN were significantly elevated in the posttreatment samples drawn at cycles 2 and 3. Although induction of HPRT mutation was only of marginal significance, results here are for the mutant frequency determination assay only. In progress is the potentially more informative analysis of the type of mutations by Southern blot and the sequencing of mutations to look for characteristic mutational spectra. The GPA assay showed a significant increase over baseline in samples drawn after cycles 3 and 4 (NO variants) and after cycles 2, 3, and 4 (NN variants). The level of GPA mutation (both NO and NN variants) was clearly elevated even 3-6 months after the last cycle of chemotherapy. Correlations were seen between HPRT and MN as well as between GPA NO and GPA NN variants. Analysis of biomarkers by treatment group does not reveal a consistent pattern or trend across all cycles.(ABSTRACT TRUNCATED AT 400 WORDS)

Adolescent↗

PubMed1984

An improved method for analysis of urinary hydroxyproline by an automated analyzer.

An improved method of analysis has been devised for estimating the amount of hydroxyproline excreted into the urine. This study has been performed to overcome the difficulties accompaning mass-examinations which are indispensable in epidimiological studies. Urine samples are pretreated with a resin-charcoal mixture for elimination of interfering substances or color inhibitions, and the supernatant is treated by evaporation to avoid complications followed by pH adjustment. The residue is reconstituted with diluted HCL. After pretreatment, samples a analyzed with an automated analyzer. Linearity of standard calibration curve, and recovery and reproducibility of this assay are satisfactory. This simple procedure of analysis requires little time. This method is also useful for routine clinical examinations.

Autoanalysis↗

PubMed1993

Preparation of fine needle aspiration biopsy samples for flow cytometric analysis.

In order to improve the quality of flow cytometric (FCM) DNA histograms, a new preparatory method was tested on samples obtained by fine needle aspiration biopsy (FNAB) of breast tumors. Twenty-four samples were obtained in vivo (group 1), and 20 were obtained from surgically resected specimens (group 2). Tumors from both groups were aspirated twice each. The first sample was injected directly into 70% ethanol, whereas the second sample was pretreated with a mixture of Tween-20 and citric acid solution (Tween-20 CA) before ethanol fixation. The coefficient of variation (CV) of G0-G1 peaks of Tween 20 CA-pretreated samples varied from 1.85 to 5.10 (mean, 3.3) in group 1 and from 1.87 to 3.72 (mean, 2.77) in group 2. The CV of G0-G1 peaks of ethanol-preserved samples ranged from 2.28 to 7.22 (mean, 5.23) in group 1 and from 1.78 to 4.04 (mean, 3.48) in group 2. The CV values of histograms obtained by the new protocol were significantly lower (group 1, P < .05; group 2, P < .01).

Analysis of Variance↗

PubMedDate not supplied

Determination of chromium in wine and other alcoholic beverages consumed in spain by electrothermal atomic absorption spectrometry.

A reliable and rapid method is described for the determination of chromium in alcoholic beverages by electrothermal atomic absorption spectrometry (ETAAS) after optimization of the temperature-time program for the graphite furnace. Chromium in wines was determined directly without sample pretreatment. Other samples were first mineralized with HNO3 and V2O5. The method was validated, and its analytical characteristics were checked. The detection limit was 1 pg (for a sample volume of 10 microL). Accuracy was 99.20 +/- 3.80% for wines and 98.90 +/- 2.00% for other beverages. Precision ranged from 3.50 to 4.50%. The proposed method was applied to 124 samples of alcoholic beverages frequently consumed in Spain. Mean values obtained (microgram/L) were 20.28 for wines, 8.75 for beer, 7.85 for cider, 14.60 for brandy, 7.50 for rum, 10.25 for whisky, 8.80 for gin, 9.25 for vodka, 24.45 for anisette, and 10.08 for liquors. The advantages of the proposed method (rapidity, sensitivity) and versatility) make it useful for routine analyses and for estimations of dietary intake of chromium.

Alcoholic Beverages↗

PubMed1999

Pharmacokinetic investigations with direct injection of plasma samples: possible savings using capillary electrophoresis (CE).

Capillary electrophoresis (CE) is often regarded as a separation technique of choice because of its high selectivity and its cost advantages compared to LC.RSD% of 0.5% have become standard for quality control assays. Using CE, sample pretreatment can often be significantly reduced, leading to notable savings of labor and reagent costs. Moreover, errors from sample pretreatment steps are avoided. A number of pharmaceuticals (e.g. acetaminophen, salicylic acid, sulfamethoxazole, theophylline, tolbutamide, and trimethoprim) have been determined in human plasma on underivatized fused silica capillaries by MEKC without sample pretreatment, the total analysis time being only 10 min. An sodium dodecyl sulfate-containing borate buffer (60 mM with 200 mM SDS) at pH 10 has been used. Between runs, proteins adsorbed to the capillary wall are removed by a rinsing regimen consisting of SDS buffer and either acetonitrile (e.g. 50% v/v) or isopropanol (e.g. 10% v/v). Other rinsing approaches are discussed (salts, enzyme containing solutions, organic solvents, sodium hydroxide, hydrofluoric acid). The separation system is tested in a concentration range between 10 ng/mL and 100 micrograms/mL, the detection limit being about 5 ng/mL. The sensitivity has been substantially improved compared to preceding work using field-amplified injection mechanisms and efficient computer algorithms that take advantage of multiwavelength detection. Correlations between the limit of quantitation (LOQ), the limit of detection (LOD) and the signal/noise ratio are discussed. A day-to-day precision for relative peak areas of 1 to 2% relsdv (n > 40) has been reached in the upper concentration range. Thus, not only drug monitoring but also pharmacokinetic investigations from blood plasma have become possible without further sample pretreatment.

Acetaminophen↗

PubMed2003

On-line SPE-CE for the determination of insulin derivatives in biological fluids.

An on-line SPE-CE system is described for the determination of insulin derivatives in urine, serum and plasma. By combining techniques based on different separation mechanisms, in this case reversed-phase SPE and CE, a more selective sample clean-up is obtained. The described on-line SPE-CE procedure is able to desalt and clean biological samples, resulting in more repeatable electrophoretic results as well as a good linearity for urine, serum and plasma samples spiked with insulin derivatives, thus proving the elimination of detrimental effects caused by the sample matrix. The on-line SPE-CE system was linear for urine, serum and plasma samples spiked with insulin derivatives between 5 and 80 mg/l. The repeatability in migration time was below 1% relative standard deviation (R.S.D.). The repeatability of the peak was better (<2.4% R.S.D.) when no off-line precipitation reaction (<6.2% R.S.D.) was used, proving the beneficial characteristics of on-line sample pretreatment procedures over off-line sample pretreatment procedures which are prone to sample losses and contamination.

Amino Acid Sequence↗

PubMed1992

Determination of substituted purines in body fluids by micellar electrokinetic capillary chromatography with direct sample injection.

Many substituted purines (theobromine, caffeine, paraxanthine, theophylline and uric acid, as well as other methylated xanthines and uric acids) can easily be separated and analysed in one run using micellar electrokinetic capillary chromatography with a boratephosphate buffer containing 75 mM sodium dodecyl sulphate (pH approximately 9). Serum, saliva and urine samples collected after the self-administration of caffeine and serum samples from patients receiving theophylline or caffeine pharmacotherapy were screened for substituted purines. The data presented show the ease of using on-column multi-wavelength detection for investigating the feasibility of direct sample application, the characterization of sample pretreatment procedures and peak confirmation by comparing absorption spectra. It is shown that the determination of purines in serum and saliva samples, including therapeutic concentrations of caffeine and theophylline, can be accomplished without any sample pretreatment, whereas sample extraction is required for the determination of purines in urine. Quantitative data for the determination of micromolar amounts of theophylline (samples from adult patients) and caffeine (samples from infants born prematurely) in serum samples compared well with data obtained by non-isotopic immunoassays. Micellar electrokinetic capillary chromatography with the direct injection of serum or saliva samples requires only microlitre volumes of sample and several different compounds can be determined within a few minutes.

Adult↗

PubMed1983

Serum protein-bound carbohydrates and small cell carcinoma of the lung. Correlations with extent of disease, tumor burden, survival, and clinical response categories.

The levels for serum protein bound neutral carbohydrates (fucose, mannose, and galactose) were determined at specific intervals for 40 patients with small cell carcinoma of the lung and compared to the corresponding carcinoembryonic antigen (CEA) levels. In pretreatment samples, the frequency of elevation was 92.5% for fucose and 77.5% each for mannose and for galactose. CEA determined in these same samples was elevated (greater than 5 ng/ml) in 45.0%. One or more of the three carbohydrate levels were elevated in pretreatment serum of 95.0% of the patients. The individual frequency of elevation for each carbohydrate was significantly related to initial stage of disease (P less than 0.01). Median survival was significantly longer for patients based on a discriminant of less than 3 carbohydrates elevated in pretreatment samples (25 months) to all 3 elevated (11 months) with P = 0.0302. A single value, termed the biomarker index, was calculated to represent the summation of the individual carbohydrate levels per individual serum sample. The biomarker index was found to be directly correlated with extent of primary disease, number of metastic sites, tumor burden, and clinical response categories assessed at serial time points. For patients with both low Biomarker Index values and normal CEA levels in pretreatment samples, an initial rise in both determinations occurred frequently corresponding to partial or complete tumor response. The occurrence of such discordant results must be considered as a likely possibility for those patients with low or normal pretreatment biological marker levels and subsequent response to primary chemotherapy.

Blood Proteins↗

PubMed2003

A high-throughput on-line microdialysis-capillary assay for D-serine.

A high-throughput method is described for the analysis of D-serine and other neurotransmitters in tissue homogenates. Analysis is performed by microdialysis-capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection in a sheath flow detection cell. Sample pretreatment is not required as microdialysis sampling excludes proteins and cell fragments. Primary amines are derivatized on-line with o-phthaldialdehyde (OPA) in the presence of beta-mercaptoethanol followed by on-line CE-LIF analysis. Under the separation conditions described here, D-serine is resolved from L-serine and other primary amines commonly found in biological samples. Each separation requires less than 22 s. Eliminating the need for sample pretreatment and performing the high-speed CE analysis on-line significantly reduces the time required for D-serine analysis when compared with traditional methods. This method has been used to quantify D-serine levels in larval tiger salamander retinal homogenates, as well as dopamine, gamma-amino-n-butyric acid (GABA), glutamate and L-aspartate. D-serine release from an intact retina was also detected.

Animals↗

PubMed1975

The interferon refractory state. II. Biological characterization of a refractoriness-inducing protein.

When L929 cells were treated with samples collected from cells or mice producing interferon, almost complete refractoriness to subsequent interferon induction was observed. The relationship of this refractoriness-inducing product (RIP) to the antiviral activity of the samples was determined. Using interferon in the pretreatment sample as a measure of RIP concentration, a semilog plot of the pretreatment interferon titer and interferon subsequently produced, resulted in an approximately linear relationship between 10 and 100 units of interferon in the pretreatment sample. This relationship was largely independent of multiplicity of Newcastle disease virus (NDV) used to produce interferon in the treated monolayers. A 2- to 6-fold temporal differentiation in the refractoriness-inducing activity and the antiviral activities was obtained both in vitro and in vivo. Supernatants collected 48 hr after NDV infection of L929 cells had 2- to 4-fold more refractoriness-inducing activity than those collected earlier. In serum of ICR mice given poly rIn:rCn-poly-D-lysine, a quantitatively similar temporal increase in refractoriness-inducing activity was observed. Treatment of L929 cells with cycloheximide resulted in a 4-fold increase in interferon production. At equivalent interferon concentrations, supernatants from the cycloheximide-treated monolayers inhibited interferon production 4-fold less than did comparable supernatants from non-cycloheximide-treated monolayers. Soluble cell extracts inhibited interferon production to a greater degree than did extracellular preparations. The protein nature of the refractoriness-inducing activity was suggested by its trypsin sensitivity. These findings suggest that the refractoriness-inducing and antiviral activities of interferon preparations result from different proteins.

Antiviral Agents↗

PubMed2005

Phenotype and frequency of Epstein-Barr virus-infected cells in pretreatment blood samples from patients with Hodgkin lymphoma.

An accumulating body of data suggests that the Epstein-Barr virus (EBV), a lymphotropic herpesvirus, is involved in the pathogenesis of a proportion of cases of Hodgkin lymphoma (HL). In this study, we showed that the frequency of circulating EBV-infected cells was significantly higher (P < 0.001) in pretreatment blood samples from EBV-associated cases when compared with non-EBV-associated cases. We further showed that in patients with EBV-associated disease, the virus persisted in the peripheral blood in memory B cells. This phenotype is consistent with that seen in healthy seropositive controls, post-transplant patients and patients with acute infectious mononucleosis. The data suggest that an increased frequency of EBV carrying B cells in peripheral blood is associated with EBV-associated HL.

Adolescent↗

PubMed2000

Capillary electrophoresis separations on a planar chip with the column-coupling configuration of the separation channels

Some basic aspects of capillary electrophoresis (CE) separations on a poly(methyl methacrylate) chip provided with two separation channels in the column-coupling (CC) configuration and on-column conductivity detectors were studied. The CE methods employed in this study included isotachophoresis (ITP), capillary zone electrophoresis (CZE), and CZE with on-line ITP sample pretreatment (ITP-CZE). Hydrodynamic and electroosmotic flows of the solution in the separation compartment of the chip were suppressed, and electrophoresis was a dominant transport process in the separations performed by these methods. Very reproducible migration velocities of the separated constituents were typical under such transport conditions, and consequently, test analytes could be quantified by various ITP techniques with 1-2% RSD. The CC configuration of the separation channels provides means for an effective combination of an enhanced load capacity of the separation system with high detection sensitivities for the analytes in concentration-cascade ITP separations. In this way, for example, succinate, acetate, and benzoate could be separated also in instances when they were present in the loaded sample (1.2 microL) at 1 mmol/L concentrations while their limits of detection ranged from 8 to 12 micromol/L concentrations. A well-defined ITP concentration of the analyte(s) combined with an in-column sample cleanup (via an electrophoretically driven removal of the matrix constituents from the separation compartment) can be integrated into the separations performed on the CC chip. These sample pretreatment capabilities were investigated in ITP-CZE separations of model samples in which nitrite, phosphate, and fluoride (each at a 10 micromol/L concentration) accompanied matrix constituents (sulfate and chloride) at considerably higher concentrations. Here, both the concentration of the analytes and cleanup of the sample were included in the ITP separation in the first separation channel while the second separation channel served for the CZE separation of the ITP pretreated sample and the detection of the analytes.

Journal Article↗

PubMed2006

Reduction of carry-over in column-switching HPLC/MS system with automated system washing procedure for highly sensitive direct analysis of donepezil in dog plasma.

To solve the problem of carry-over--a persistent chromatographic challenge for bioanalytical assays with highly sensitive detector such as a mass spectrometry (MS)--a new on-line sample pretreatment HPLC/MS system using a column-switching technique was established. This system was designed to reduce carry-over based on a hydrophobic interaction mechanism using a washing function (multi-mobile phase flow system), as well as to remove impurities on-line in a mobile phase for the pretreatment. As a result, a washing function in this system was enabled to reduce carry-over and to remove impurities in a mobile phase by automatic operation. Therefore, concentration levels of donepezil (DH) in dog plasma as low as 10 pg/mL could be determined using this on-line sample pretreatment HPLC/MS system. In addition, method validation results of specificity, linearity, accuracy, precision, lower limit of quantitation (LLOQ), and carry-over demonstrated that this on-line sample pretreatment HPLC/MS system was robust and valid as a practical assay of drugs and metabolites in the biological samples.

Animals↗

PubMed1994

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 beta-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 beta-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 beta-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 microgram/kg (ppb).

Adrenergic beta-Agonists↗