Development of a colloidal gold-based lateral-flow immunoassay for the rapid simultaneous detection of clenbuterol and ractopamine in swine urine

A multianalyte lateral-flow immunochromatographic technique using colloidal gold-labeled polyclonal antibodies was developed for the rapid simultaneous detection of clenbuterol and ractopamine. The assay procedure could be accomplished within 5 min, and the results of this qualitative one-step assay were evaluated visually according to whether test lines appeared or not. When applied to the swine urines, the detection limit and the half maximal inhibitory concentration (IC₅₀) of the test strip under an optical density scanner were calculated to be 0.1 ± 0.01 ng mL⁻¹ and 0.1 ± 0.01 ng mL⁻¹, 0.56 ± 0.08 ng mL⁻¹, and 0.71 ± 0.06 ng mL⁻¹, respectively, the cut-off levels with the naked eye of 1 ng mL⁻¹ and 1 ng mL⁻¹ for clenbuterol and ractopamine were observed. Parallel analysis of swine urine samples with clenbuterol and ractopamine showed comparable results obtained from the multianalyte lateral-flow test strip and GC-MS. Therefore, the described multianalyte lateral-flow test strip can be used as a reliable, rapid, and cost-effective on-site screening technique for the simultaneous determination of clenbuterol and ractopamine residues in swine urine.      

A rapid in vitro assay of cobalamin in human urine and medical tablets using ICP-MS

A rapid and simple as well as sensitive inductively coupled plasma mass spectrometry (ICP-MS) method for the determination of cobalamin is described. Cobalamin in human urine and medicine tablet solutions was converted on-line into free cobalt ions in acid medium, the cobalt ions were then detected by ICP-MS. Cobalamin was determined by measuring the increase of integral counts per second intensity, which was linear over the cobalamin concentration range of 1.0 × 10⁻¹⁰ g mL⁻¹ to 8.0 × 10⁻⁵ g mL⁻¹, and the limit of detection was 0.05 ng mL⁻¹ (3σ). At the pump rate of 30 rotations per minute, one analysis cycle of cobalamin, including sampling and washing, could be accomplished in 0.5 min with the relative standard deviations of less than 5 %. The proposed procedure was applied successfully in monitoring cobalamin in human urine without any pretreatment process and in rapid determination of cobalamin in multivitamin tablets.     

Analysis of 5-hydroxy-N-methyl-2-pyrrolidone and 2-hydroxy-N-methylsuccinimide in plasma

Summary A method for the determination of 5-hydroxy-N-methyl-2-pyrrolidone (5-HNMP) and 2-hydroxy-N-methylsuccinimide (2-HMSI) in plasma was developed. 5-HNMP and 2-HMSI are metabolites to the widely used organic solvent N-methyl-2pyrrolidone (NMP). The 5-HNMP and 2-HMSI were purified from plasma by C8 solid phase extraction, derivatised by bistrimethylsilyl trifluoroacetamid, and analysed by gas chromatography with mass spectrometric detection. For 5-HNMP, the precision was 2–7 % (120 and 780 ng mL⁻¹) and the detection limit was 6 ng mL⁻¹ (m/z 98). For 2-HMSI, the precision was 2–9 % (160 and 1000 ng mL⁻¹) and the detection limit was 4 ng mL⁻¹ (m/z 144). The method is applicable for analysis of plasma samples from workers exposed to NMP.     

Quantitative analysis of temazepam in plasma by capillary gas chromatography: Application to pharmacokinetic studies in rats

Summary A sensitive method was developed for the determination of temazepam in plasma using capillary gas chromatography. After the extraction into dichloromethane-pentane (1∶1), temazepam was quantitated as its O-trimethylsilyl derivative on a capillary column with a⁶³Ni electron capture detector using prazepam as internal standard. The detector response was found to be linear in the concentration range 0.031 to 8 μg mL⁻¹. The detection limit was about 3.5 ng mL⁻¹. The intraday and inter-day coefficients of variation were below 9%. The method was used to determine the pharmacokinetic profile of temazepam in rats after intravenous administration.     

Detection of Stanozolol and Its Major Metabolites in Human Urine by Liquid Chromatography-Tandem Mass Spectrometry

The determination of stanozolol and its metabolites in human urine has been of particular interest in sports drug testing due to its frequently revealed misuse. A simple and rapid sample preparation procedure based on consecutive solid-phase and liquid–liquid extraction with subsequent re-extraction followed by liquid chromatography and electrospray ionization tandem mass spectrometry was established. It allowed the determination of stanozolol and its metabolic products 16β-OH-stanozolol and 4β-OH-stanozolol in human urine at detection limits of 0.1, 0.2 and 0.2 ng mL⁻¹, respectively, with recoveries ranging from 5 to 38%. The robust nature of the assay and the efficient removal of interfering biological matrix provides excellent signal-to-noise ratios, and, thus, a rapid alternative to established procedures utilizing multiple solid-phase extraction or immunoaffinity chromatography strategies. More than 15 doping control urine specimens tested positive for stanozolol during the last 12 months have been confirmed using the described approach.     

Determination of tebufenpyrad and oxadiazon by solid-phase microextraction and gas chromatography-mass spectrometry

Summary A method for determination of trace amounts of the pesticides tebufenpyrad and oxadiazon, previous solid-phase microextraction (SPME), was developed using gas chromatographymass spectrometry and selected ion monitoring (GC-MS; SIM). Both pesticides were extracted with a fused silica fiber coated with 100 μm polydimethylsiloxane. The effects of pH ionic strength, sample volume, extraction and desorption times as well as extraction temperature were studied. The linear concentration range of application was 0.5–250 ng mL⁻¹ for both compounds, with a detection limit of 0.06 ng mL⁻¹ for tebufenpyrad and 0.02 ng mL⁻¹ for oxadiazon. SPME-GC-MS analysis yielded good reproducibility (RSD between 7.5–10.1%). It was used to check the eventual existence of tebufenpyrad and oxadiazon above this limit in water and soil samples from Granada (Spain) as well as in human urine samples. The method validation was completed with spiked matrix samples. It can be applied as a monitoring tool for water, soil and urine in the investigation of environmental and occupational exposure to tebufenpyrad and oxadiazon.     

Development and validation of an HPLC method for the simultaneous determination of clozapine and desmethylclozapine in plasma of schizophrenic patients

Summary A high-performance liquid chromatographic method with amperometric detection has been developed for the determination of levels of clozapine (CLZ) and its active metabolite N-desmethylclozapine (DMC) in human plasma. The analysis was performed on a 5 μm C8 reversed phase column (150×4.6 mm i.d.), with acetonitrile-phosphate buffer (pH 3.5), as the mobile phase. The detection voltage was +800 mV and the cell and column temperature were 50°C. Linear responses were obtained between 2 ng mL⁻¹ and 100 ng mL⁻¹. Absolute recovery for both clozapine and desmethylclozapine exceeded 88% and the detection limit was 1 ng mL⁻¹. Repeatability, intermediate precision and accuracy were satisfactory. The method, which is rapid, sensitive and selective, has been applied to therapeutic drug monitoring in schizophrenic patients following administration of Leponex^? tablets. In 21 patients in steady state at a mean daily clozapine dosage of 358 mg (ranging from 150 to 500 mg day⁻¹), clozapine levels averaged 379 ng mL⁻¹ (ranging from 102 to 818 ng mL⁻¹) and DMC levels averaged 233 ng mL⁻¹ (ranging from 70 to 540 ng mL⁻¹). The method requires only a very small amount of plasma (100 μL), and thus it is suitable for pharmacokinetic studies, as well as for therapeutic drug monitoring.     

Sensitive determination of 3-methoxy-4-hydroxyphenylglycol (MHPG) in human plasma by HPLC with electrochemical detection

Summary This study deals with the development of a new HPLC method for the determination of 3-methoxy-4-hydroxyphenylglycol (MHPG), the main noradrenaline metabolite in human plasma. A Varian reversed-phase column (C₈; 250 mm×4.6 mm i.d.; 5 μm particles) was used as the stationary phase and an aqueous solution of citric acid, 1-octanesulfonic acid, EDTA, and methanol was used as the mobile phase. Coulometric electrochemical detection (ED) was used to obtain the highest sensitivity. Isolation of MHPG from plasma was accomplished by means of a new solid-phase extraction procedure after a protein precipitation step. The extraction yield of MHPG from plasma was very high (>97%). Linearity was observed in the 0.5–25 ng mL⁻¹ concentration range; the limit of detection was 0.2 ng mL⁻¹ and the limit of quantitation was 0.5 ng mL⁻¹. Repeatability (RSD,%) for plasma samples was found to be <3.2% and intermediate precision was <4.3%. The method was applied to the determination of MHPG in the plasma of healthy subjects under experimentally-induced psychological stress.     

LC–ESI–MS for Rapid and Sensitive Determination of Aripiprazole in Human Plasma

A rapid, sensitive, and accurate high-performance liquid-chromatographic–mass spectrometric (HPLC–MS) method, with estazolam as internal standard, has been developed and validated for determination of aripiprazole in human plasma. After liquid–liquid extraction the compound was analyzed by HPLC on a C₁₈ column, with acetonitrile—30 mm ammonium acetate containing 0.1% formic acid, 58:42 (v/v), as mobile phase, coupled with electrospray ionization mass spectrometry (ESI-MS). The protonated analyte was quantified by selected-ion recording (SIR) with a quadrupole mass spectrometer in positive-ion mode. Calibration plots were linear over the concentration range 19.9–1119.6 ng mL⁻¹. Intra-day and inter-day precision (CV%) and accuracy (RE%) for quality-control samples (37.3, 124.4, and 622.0 ng mL⁻¹) ranged between 2.5 and 9.0% and between 1.3 and 3.5%, respectively. Extraction recovery of aripiprazole from plasma was in the range 75.8–84.1%. The method enables rapid, sensitive, precise, and accurate measurement of the concentration of aripiprazole in human plasma.     

Determination of olanzapine and desmethylolanzapine in the plasma of schizophrenic patients by means of an improved HPLC method with amperometric detection

Summary An improved HPLC method with electrochemical detection has been developed for the determination of olanzapine and its main metabolite, desmethylolanzapine, in human plasma. Chromatographic separation and analysis were performed on a C₈ reversed-phase column with a mixture of methanol, acetonitrile, and pH 3.7 phosphate buffer as mobile phase; 2-methylolanzapine was used as internal standard. Careful pretreatment of the plasma samples was implemented by means of solid phase extraction (SPE). Response was linearly dependent on concentration and precision was satisfactory over the concentration range 0.5–75.0 ng mL⁻¹ for both analytes. The limit of detection was 0.2 ng mL⁻¹ for both analytes. Application to plasma samples of patients treated with Zyprexa tablets gave good results. Because of its sensitivity and selectivity, and the need for small plasma samples, this method seems to be a useful tool for clinical monitoring.     

Determination of Eprinomectin in Bovine Urine and Feces Using HPLC with Fluorescence Detection

Eprinomectin is a novel and potent antiparasitic animal health drug. An analytical procedure for the determination of EPR in bovine urine and feces has been developed. The urine sample was centrifuged and alkalized with ammonia following solid phase extraction. The fecal sample was extracted with acetonitrile, defatted with hexane, cleaned-up using C18 cartridge. All samples were analyzed by high performance liquid chromatography with fluorescence detection after derivatization with N-methylimidazole. The limits of detection are 0.5 ng mL⁻¹ and 0.5 ng g⁻¹, respectively. Fortified at 2, 10, 50, and 100 ng mL⁻¹(ng g⁻¹), inter-assay recoveries of EPR in cattle urine and feces were in the range of 87.9–91.5% and 78.6–86.3%, with coefficients of variation of 5.4–10.2% and 1.4–7.2%, respectively. Intra-assay mean recoveries of the analytes were 82.2–86.5% and 79.6–87.3%, with coefficients of variation of 7.8–11.5% and 6.3–7.8%, respectively. The method was used to study the excretion of eprinomectin in bovine urine and feces after subcutaneous administration at a dose of 0.5 mg kg⁻¹.     

Determination of eight penicillins in serum from cattle and pigs by generic HPLC method

Summary An HPLC method was developed for determination of amoxicillin, penicillin G, penicillin V, ampicillin, oxacillin, cloxacillin, nafcillin and dicloxacillin in serum from pigs and cattle. Serum was cleaned up by solid-phase extraction (SPE), ultra-filtered and derivatised. The method was linear in the range tested up to 2000 ng mL⁻¹ of individual penicillins in serum. Limits of detection were 11–14 ng mL⁻¹. Mean recoveries were 90–103% in the range 20–2000 ng mL⁻¹. The relative repeatability, standard deviation was <10% at 20 ng mL⁻¹ level and <6% in the range 100–2000 ng mL⁻¹.     

Determination of Antioxidants and Preservatives in Cosmetics by SPME Combined with GC–MS

A rapid and simple procedure for the determination of antioxidants and preservatives in cosmetics has been developed utilizing solid-phase microextraction combined with GC–MS. A silica fiber coated with polyacrylate provided the highest extraction efficiency. Detection limits in the range from 0.4 to 8.5 ng mL⁻¹ were obtained. Linearity is over a wide range from 1 to 2,000 ng mL⁻¹ with a relative standard deviation under 16%. Cosmetic from a local supermarket were analysed for antioxidants and preservatives to demonstrate the effectiveness of the proposed method. The concentration of antioxidants and preservatives determined was 20–1,218 μg g⁻¹ for methylparaben and 5–3,779 μg g⁻¹ for propylparaben.     

Determination and Pharmacokinetic Study of 10-O-Dimethylaminoethylginkgolide B in Rat Plasma by LC–MS

To evaluate the pharmacokinetics of a novel analogue of ginkgolide B, 10-O-dimethylaminoethylginkgolide B (XQ-1) in rat plasma in pre-clinical studies, a sensitive and specific liquid chromatographic method with electrospray ionization mass spectrometry detection (LC–ESI–MS) was developed and validated. After a simple extraction with ethyl acetate, XQ-1 was analyzed on a Shim-pack C18 column with a mobile phase of a mixture of 1 μmol L⁻¹ ammonium acetate containing 0.02% formic acid and methanol (55:45, v/v) at a flowrate of 0.3 mL min⁻¹. Detection was performed in selected ion monitoring (SIM) mode using target ions at [M + H]⁺ m/z 496.05 for XQ-1 and m/z 432.10 for the internal standard (lafutidine). Linearity was established for the concentration range from 2 to 1,000 ng mL⁻¹ . The extraction recoveries ranged from 86.0 to 89.9% in plasma at concentrations of 5, 50, and 500 ng mL⁻¹. The lower limit of quantification was 2 ng mL⁻¹ with 100 μL plasma. The validated method was successfully applied to a pharmacokinetic study after intragastic administration of XQ-1 mesylate in rats at a dose of 20 mg kg⁻¹.     

High performance liquid chromatography method for the determination of meropenem in human plasma

Summary This paper describes an HPLC method for the determination of meropenem in human plasma. The method uses solid phase extraction (SPE) of the samples and has good sensitivity, precision and accuracy. The limit of quantification in plasma samples is 0.02 μg mL⁻¹. Calibration curves were linear over a large dynamic range, namely within 0.02–50 μg mL⁻¹. The method was applied to the determination of meropenem levels in patients receiving meropenem, as a single dose or at steady state.     

Automated high-performance liquid chromatographic determination of amphetamine in biological fluids using column-switching and on-column derivatization

Summary A rapid and simple liquid-chromatographic method has been developed for on-line quantification of amphetamine in biological fluids. Untreated samples (20 μL) are injected directly into the chromatographic system and purified on a 20 mm×2.1 mm i.d. pre-column packed with 30 μm Hypersil C₁₈ stationary phase. After clean-up the analyte is transferred to the analytical column (125 mm×4 mm i.d., 5 μm LiChrospher 100 RP₁₈) for derivatization and separation using a mixture of acetonitrile and the derivatization reagent (o-phthaldialdehyde andN-acetyl-L-cysteine) as the mobile phase. The experimental conditions for on-line derivatization and resolution of the amphetamine have been optimized, and the results have been compared with those obtained by derivatizing the analyte in pre-column mode. The method described has been applied to the determination of amphetamine in plasma and urine. Good linearity and reproducibility were obtained in the 0.1–10.0 μg mL⁻¹ concentration range, and limits of detection were 25 ng mL⁻¹ and 10 ng mL⁻¹ with UV and fluorescence detection, respectively. The procedure described is very simple and rapid, because no off-line manipulation of the sample is required; the total analysis time is approximately 8 min.     

Optimization of solid-phase microextraction procedures for the determination of tricyclic antidepressants and anticonvulsants in plasma samples by liquid chromatography

Simple, sensitive, and reproducible off-line solid-phase microextraction and liquid chromatography (SPME/LC) methods are described for the determination of seven anticonvulsants and tricyclic antidepressants in human plasma. Factorial design and simplex methodology were applied in the optimization of the SPME procedure for tricyclic antidepressants analyses. Important factors in the SPME efficiency are discussed, such as the fiber coatings (both lab-made and commercial), extraction time, pH, ionic strength, influence of plasma proteins, and desorption conditions. The development of the lab-made fiber coatings, namely, octadecylsilane, aminosilane, and polyurethane, are further described and applied to anticonvulsants analyses. The investigated plasmatic range for the evaluated anticonvulsants, using CW-TPR fiber, were the following: phenylethylmalonamide (3.00–40.0 μg mL⁻¹), phenobarbital (5.00–40.0 μg mL⁻¹), primidone (3.00–40.0 μg mL⁻¹), carbamazepine and carbamazepine-epoxide (2.00–24.0 μg mL⁻¹), phenytoin (2.00–40.0 μg mL⁻¹), and lamotrigine (0.50–12.0 μg mL⁻¹). The antidepressants’ linear plasmatic concentration ranged from 75.0 to 500 ng mL⁻¹ for imipramine, amitriptyline, and desipramine, and from 50.0 to 500 ng mL⁻¹ for nortriptyline, being in all cases, the limit of quantification represented by the lowest value. The precision (interassays) for all investigated drugs in plasma sample spiked with different concentrations of each analyte and submitted to the described procedures were lower than 15%. The off-line SPME/LC methodologies developed allow anticonvulsants and antidepressants analyses from therapeutic to toxic levels for therapeutic drug monitoring.     

Resonance Rayleigh scattering spectra of tetracycline antibiotic–Cu(II)–titan yellow systems and their applications in analytical chemistry

Tetracycline antibiotics (TCs) such as doxycycline (DOTC), chlortetracycline (CTC), oxytetracycline (OTC), and tetracycline (TC) react with Cu(II) in pH 3.5 BR buffer medium to form 1:1 cationic chelates, which further react with titan yellow to form 2:1 ion association complexes. These result in great enhancement of resonance Rayleigh scattering (RRS) and the appearance of new RRS spectra. The ion association complexes of DOTC, CTC, OTC, and TC have similar spectral characteristics and their maximum RRS wavelengths are all located at 464 nm. The quantitative determination ranges and the detection limits (3σ) of the four TCs are 0.037–4.8 μg mL⁻¹ and 11.2 ng mL⁻¹ for DOTC, 0.041–5.2 μg mL⁻¹ and 12.4 ng mL⁻¹ for CTC, 0.050–4.8 μg mL⁻¹ and 15.1 ng mL⁻¹ for TC, and 0.088–5.0 μg mL⁻¹ and 26.3 ng mL⁻¹ for OTC, respectively. The optimum reaction conditions, the effects of foreign substances, the structure of ternary complexes, and the reaction mechanism are discussed. A sensitive, rapid, and simple RRS method for the determination of DOTC has been developed.     

Solid-phase microextraction coupled with microcolumn liquid chromatography for the analysis of amitriptyline in human urine

Summary Solid-phase microextraction (SPME) is a solvent-free sample-preparation technique that enables isolation and pre-concentration of analytes from a sample on a thin film coating a fused-silica fiber. In this study SPME coupled with microcolumn liquid chromatography (micro LC) has been used for the determination of four tricyclic antidepressants (amitriptyline, imipramine, nortriptyline, and desipramine) in human urine. SPME conditions which affect extraction efficiency were optimized, and under the optimum conditions the system was a few hundred times more sensitive than direct LC analysis without SPME. For amitriptyline the detection limit was 3 ng mL⁻¹ and the calibration curve was linear in the range of 5–500 ng mL⁻¹. The SPME-micro LC method has been applied to the analysis of amitriptyline in patient’s urine.     

A high-performance liquid-chromatographic method for the determination of ochratoxin a in human plasma

Summary A high-performance liquid-chromatographic method is described for the quantitative determination of the mycotoxin ochratoxin A (OTA) in human plasma. The assay involves extraction with chloroform and sodium bicarbonate then HPLC with fluorescence detection. The method was validated in terms of selectivity, recovery, linearity, precision (within-day and between-day variability), accuracy, detection and quantification limits, and the stability of OTA in plasma and treated samples. The limit of detection was 0.4 ng mL⁻¹ of OTA in methanol, corresponding to 0.52 ng ml⁻¹ OTA in plasma. This assay was successfully applied for the determination of OTA levels in human plasma.