Single-dose administration of clenbuterol is detectable in dried blood spots.

Clenbuterol is a ß2 -agonist prescribed for asthmatic patients in some countries. Based on its anabolic and lipolytic effects observed in studies on rodents and in livestock destined for food production, clenbuterol is abused by bodybuilders and athletes seeking leanness. Urinary clenbuterol analysis is part of routine doping analysis. However, the collection of urine samples is time-consuming and can be intimidating for athletes. Dried blood spot (DBS) appears attractive as an alternative matrix, but the detectability of clenbuterol in humans through DBS has not been investigated. This study evaluated if clenbuterol could be detected in DBS and urine collected from six healthy men after oral intake of 80 µg clenbuterol. The DBS and urine samples were collected at 0, 3, 8, 24, and 72 h post-ingestion, with additional urine collections on days 7 and 10. Using LC-MS/MS, it was shown that clenbuterol could be detected in all DBS samples for 24 h post-ingestion and with 50% sensitivity 3 days after ingestion. The DBS method was 100% specific. Evaluation of analyte stability showed that clenbuterol is stable in DBS for at least 365 days at room temperature when using desiccant and avoiding light exposure. In urine, clenbuterol was detectable for at least 7-10 days after ingestion. Urinary clenbuterol concentrations below 5 ng/mL were present in some subjects 24 h after administration. Collectively, these data indicate that DBS are suitable for routine doping control analysis of clenbuterol with a detection window of at least 3 days after oral administration of 80 µg.

Enantiomeric analysis of clenbuterol in Chinese people by LC-MS/MS to distinguish doping abuse from meat contamination.

Aim: Follow-up investigations are often required for clenbuterol-positive cases. A method to distinguish doping abuse from meat contamination was developed. Materials & methods: A total of 26 volunteers were recruited to ingest clenbuterol contaminated-pork and clenbuterol tablets. Results: For 20 volunteers, after ingestion of contaminated-pork, R-(-)/S-(+)-clenbuterol ratio was <1.0, while the value was >1.0 after taking clenbuterol tablets. However, after taking clenbuterol tablets, some ratio points of the other six volunteers were between 0.9 and 1.0. A case of an abnormal cold and fever, which returned to normal after recovery, was also reported firstly. Conclusion: A change in R-(-)/S-(+)-clenbuterol was reported in the Chinese population initially. A ratio of 0.9 was recommended in doping related cases for the Chinese population.

Clenbuterol: a new toxic substance in paediatrics.

A 13-year-old girl presented to the emergency department with acute onset of chest pain, nausea and tremor. The patient denied drug ingestion, and urine toxicology was negative. ECG demonstrated sinus tachycardia, prolonged QTc (541 ms) and ST depression. Laboratory testing demonstrated metabolic acidosis, hypokalaemia, hypophosphataemia and hyperglycaemia. She was commenced on continuous cardiac monitoring and treated with intravenous fluids and electrolyte replacement. Presenting features and laboratory abnormalities resolved within 48 hours. The National Poisons Information Service and Clinical Biochemistry were integral to her management, advising the clinical team on the likeliest aetiology. Five weeks after discharge, urine toxicology, using mass spectrometry, identified clenbuterol. Clenbuterol is an oral ß2-agonist with anabolic and lipolytic effects that is misused as a performance and image enhancing drug. Clinicians must be aware of the increasing availability of these drugs and their potential for causing harm in children and adolescents.

Selective extraction and detection of ß-agonists in swine urine for monitoring illegal use in livestock breeding.

The illegal use of ß-agonists often endangers animal-derived food safety. In this study, a selective detection method for ß-agonists in swine urine was established via the combination of polymeric ionic liquid-molecularly imprinted graphene oxide-miniaturized pipette tip solid-phase extraction and high-performance liquid chromatography. It is worth noting that this method relied mainly on the designed adsorbent, which presented a rich adsorption mechanism, fast mass transfer rate, and high selectivity, and was successfully utilized in the selective extraction of ß-agonists from swine urine samples. The proposed method has low LOD (0.20-0.56 ng/mL), high recovery (94.9-107.9%), and high reusability (4 times, 91.9-108.8%), which indicates its high potential as a selective, sensitive, accurate, and nonfatal method for monitoring the illegal use of ß-agonists in the livestock breeding stage.

Simultaneous Detection of Multiple ß-Adrenergic Agonists with 2-Directional Lateral Flow Strip Platform.

Clenbuterol (CL), salbutamol (SAL) and ractopamine (RAC) are the three common ß-adrenergic agonists, which are the main hazards in food safety and affect human health through the food chain. A convenient and efficient method is urgently required to perform on-site detection of multiple ß-adrenergic agonists to avoid frequent poisoning incidents. In this paper, a 2-directional lateral flow strip technique (2-directional LFS) is developed for rapid and simultaneous detection of CL, SAL and RAC with single sampling. Compared to the conventional lateral flow strip, this 2-directional LFS technique can realize simultaneous detection of three or more target analytes without any change of intrinsic simplicity of LFS. Furthermore, this 2-directional LFS can effectively avoid the potential intrinsic cross-reactivity among the reagents to analogues. Under the optimized conditions, CL, SAL and RAC were all successfully determined with satisfactory results in both buffer and urine samples with the detection limit as low as 0.5 ng/mL. This 2-directional LFS technique can revolutionize the commercial single-analyte LFS products and can effectively widen the applications of the classic LFS in various fields.

Quantification and Stereochemical Composition of R-(-) and S-(+)-Clenbuterol Enantiomers in Bovine Urine by Liquid Chromatography-Tandem Mass Spectrometry.

Clenbuterol (4-amino-α-[(tert-butylamino)methyl]-3,5-dichlorobenzylalcohol) is a ß2-adrenergic agonist. The consumption of meat contaminated with clenbuterol can lead to increased heart rate, blood pressure, anxiety, palpitations and skeletal muscle tremors. Several analytical methods have been developed to identify and quantify clenbuterol in different biological matrices. In this report, we have developed a specific and sensitive analytical method for quantifying clenbuterol and performed an in-depth enantiomeric analysis in bovine urine. The method was evaluated in accordance with international guidelines, and we used an isotopically labeled analog as an internal standard. The extraction efficiency for clenbuterol in bovine urine was > 98%, the limit of detection was 0.05 ng/mL and the limit of quantification was 0.10 ng/mL. Our assay showed high specificity, no carryover was observed and the assay was linear in the range 0.10-8.0 ng/mL. Fifteen bovine urine samples were analyzed (containing clenbuterol), and an enantiomeric analysis was performed. The clenbuterol concentration range was 0.10-10.56 ng/mL across these samples. The levorotatory enantiomer was detected at greater concentrations than the dextrorotatory enantiomer, the ratio being 1.7 ± 0.6 (n = 15), and a statistical difference was observed (P < 0.05) using the Wilcoxon test.

In situ Preparation of HNbMoO6/C Nanocomposite for Sensitive Detection of Clenbuterol.

In this paper, we synthesized HNbMoO6/C composite through the calcination of octylamine-intercalated HNbMoO6 precursor. The resulting HNbMoO6/C composite showed some new phases of MoO2, MoO3, NbO2, Nb2O5, and carbon, which was fully confirmed via powder X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), selected area electron diffraction (SAED), and X-ray photoelectron spectroscopy (XPS) technologies. Besides, the HNbMoO6/C hybrid was coated on glass carbon electrode to construct an electrochemical sensor for sensitive determination of clenbuterol. The electrochemical behaviors of clenbuterol on the prepared electrode were tested by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) analysis. The results showed that the intercalated carbon can act as active sites to accelerate electron transfer. In addition, more exposed surface areas of the HNbMoO6/C composite will facilitate the electrolyte to permeate. The oxidation peak current of clenbuterol was linearly related to its concentration in the range of 1.04 × 10-5 to 7.51 × 10-4 mol L-1, and the determination limit was calculated to be 3.03 × 10-6 mol L-1 (S/N = 3). This sensor exhibits excellent stability, reproducibility, specificity, and good recoveries when applied to monitor clenbuterol in real samples.

Evaluation of R- (-) and S- (+) Clenbuterol enantiomers during a doping cycle or continuous ingestion of contaminated meat using chiral liquid chromatography by LC-TQ-MS.

Clenbuterol is known to improve competition resistance and muscular growth in athletes. Although it is an illegal drug, its use by farmers is widely spread to induce growth of their cattle. Thus, when clenbuterol is found in the urine of an athlete, there is doubt whether it was consumed with doping purposes or if it is due to the consumption of meat from a clenbuterol-fed animal. Previous studies suggest that enantiomeric relationship of clenbuterol may be different according to the intake source. However, the enantiomeric relationship throughout a doping cycle or a continuous intake of contaminated meat has not yet been explored. In this first approximation, our aim was the development and validation of a sensitive and rapid method for the determination of S- (+) and R- (─) clenbuterol enantiomers to be used in a controlled study in rats fed for one week with contaminated meat or simulating a doping cycle. Enantiomers were measured using liquid chromatography coupled to mass spectrometry with a triple quadrupole analyzer (LC-TQ-MS) and were separated on an AGP Chiralpak column. The method was fully validated following the VICH (Veterinary International Conference on Harmonization guidelines) and was linear in the range of 12.5-800 pg/mL with a correlation coefficient of ≥0.98 for each enantiomer, and with a limit of quantitation and detection (LOQ and LOD) of 12.5 pg/mL and 6.5 pg/mL, respectively, for both enantiomers. The application of this method pointed out the shift of the enantiomeric relationship in urine from rats during the first five days of the doping cycle compared to those fed with contaminated meat. This finding can be of substantial importance in further doping studies.

Immunochromatographic fluorometric determination of clenbuterol with enhanced sensitivity.

A method is described to enhance the sensitivity of an immunochromatographic assay for clenbuterol (CLE) by making use of dually-labeled gold nanoparticles (GNPs), background fluorescence blocking, and immunomagnetic separation. The GNPs were labeled with biotinylated antibody and streptavidin, respectively, and dually labeled GNPs were obtained via the biotin-streptavidin interaction to amplify the detection signal. The fluorescent signal was blocked by dually labeled GNPs and decreased as the dually labeled GNPs aggregation increases on nitrocellulose membrane, which derived from fluorescent polyvinylchloride card. However, fluorescence (measured at excitation/emission wavelengths of 518/580 nm) recovers when CLE reacts with dually labeled GNPs. Immunomagnetic separation was first applied for sample pretreatment. This can offset the matrix effect and improves the sensitivity and accuracy of the assay. Under the optimal conditions, the limits of detection of CLE visually were 0.25 µg·L-1. In addition, clenbuterol can be quantified in swine urine with a 0.03 µg·L-1 detection limit. This is 60-fold lower than current immunochromatography. Response is linear in the 0.06-0.59 µg·L-1 concentration range, and the recoveries from spiked swine urine range from 81 to 115%." Graphical abstract Schematic presentation of the strategies for improving sensitivity of immunochromatographic assay. It includes immunomagnetic separations, dually-labeled gold nanoparticles and background fluorescence blocking. The assay was applied to detect clenbuterol (CLE) in swine urine with an excellent performance.

Sensitive and Matrix-Tolerant Lateral Flow Immunoassay Based on Fluorescent Magnetic Nanobeads for the Detection of Clenbuterol in Swine Urine.

The lack of sensitivity and poor matrix tolerance are the main bottlenecks of the lateral flow immunoassay (LFIA). Here, a sensitive and matrix-tolerant method that integrated immunomagnetic separation and fluorescent lateral flow immunoassay (IMS-FLFIA) based on fluorescent magnetic nanobeads was developed to detect the clenbuterol (CLE) residue in swine urine. The limit of detection (LOD) of IMS-FLFIA is 4 times lower than that of traditional colloidal gold LFIA. This method, which exhibits similar LOD and linearity range in both phosphate-buffered saline and urine swine, is highly correlated with liquid chromatography-tandem mass spectrometry for the detection of real swine urine samples. The result indicated that IMS-FLFIA has a universal resistance to the swine urine matrix. The merits of this assay, high sensitivity, matrix tolerance, accuracy, and specificity, ensure a promising future in detection of veterinary drug residues.

The use of hair as a long-term indicator of low-dose ß2 agonist treatments in cattle: Implications for growth-promoting purposes monitoring.

The objective of this study was to assess the feasibility of using hair as a long-term indicator of cocktail (low-dose ß2 agonists) treatments in cattle. Six male Simmental cattle were treated with a mixture of low-dose clenbuterol, ractopamine, and salbutamol at dosages of 5.3, 223.3, and 50.0 µg/kg, respectively. The trial lasted for 112 days and included 28 days of treatment and 84 days of withdrawal. Plasma and urine samples taken during the treatment period contained the highest residues, with maximum concentrations of clenbuterol, ractopamine, and salbutamol in plasma of 1.49 ng/mL (Day 21), 43.78 (Day 14) ng/mL, and 8.07 ng/mL (Day 7), respectively, and in urine of 62.40 ng/mL (Day 28), 3995.77 ng/mL (Day 28), and 503.72 ng/mL (Day 1), respectively. On day 42 of withdrawal, the residues of all three ß2 agonists in plasma were below the limit of quantification (LOQ; 0.3 ng/mL for clenbuterol, and 0.5 ng/mL for ractopamine and salbutamol), and in urine samples were below or near the LOQ (the highest being ractopamine at 1.10 ng/mL). The highest concentrations of clenbuterol, ractopamine, and salbutamol in hair were 88.36, 1351.92, and 100.58 ng/g, respectively, on day 14 of withdrawal; and the residues were long-lasting, with 7.64, 28.55, and 8.77 ng/g, respectively, on day 84 of withdrawal. The results of this study demonstrate that hair could be utilized as a long-term indicator of the use of a combination of low-dose ß2 agonists in cattle, which could have implications for growth-promoting purposes monitoring.

Highly luminescent green-emitting Au nanocluster-based multiplex lateral flow immunoassay for ultrasensitive detection of clenbuterol and ractopamine.

A multiplex lateral flow immunoassay sensor based on highly luminescent green-emitting Au nanoclusters (AuNCs-MLFIA sensor) was successfully established for the simultaneous and quantitative determination of clenbuterol (Clen) and ractopamine (RAC) in swine urine. The antigens of Clen and RAC were dispersed on a nitrocellulose membrane as two test lines, and the Au nanoclusters were synthesized from 6-aza-2-thiothymine and l-arginine to obtain highly green luminescence and ultra-small nanoparticles (Arg/ATT/AuNCs). Free carboxyl groups on Arg/ATT/AuNCs enabled conjugation with biomolecules to afford an indicator for the biosensor. The AuNCs-MLFIA sensor is based on the indirect competition assay and could successfully detect samples within 18 min without sample pretreatment, qualitative results can be obtained by visual inspection under a UV lamp. The limits of detection of Clen and RAC by the naked eye were both 0.25 µg L-1. In addition, the AuNCs-MLFIA sensor allowed quantitative detection combined with a portable fluorescence reader. The half-maximal inhibitory concentrations of Clen and RAC were 0.06 and 0.32 µg L-1, respectively, with detection limits of 0.003 and 0.023 µg L-1. Thirty blind-spiked swine urine samples were analyzed by the AuNCs-MLFIA sensor and liquid chromatography-tandem mass spectrometry, and the results of the two methods showed a significant correlation. The newly developed AuNCs-MLFIA sensor overcomes several limitations of conventional LFIA sensors, including their low sensitivity, limitation to quantify analytes, and single-analyte detection.

A fluorometric clenbuterol immunoassay based on the use of organic/inorganic hybrid nanoflowers modified with gold nanoclusters and artificial antigen.

Organic/inorganic hybrid nanoflowers were synthesized from calcium phosphate and protein modified fluorescent gold nanoclusters and antigens. These nanoflowers are shown to be well suited labels for bioassay because they fulfill the functions of biological recognition and signal output. A fluorometric immunoassay was developed that was combined with immunomagnetic separation. In the detection system, the red fluorescence of the supernatant (measured at excitation/emission wavelengths of 360/640 nm) is found to be proportional to the clenbuterol (Clen) concentration after two immunomagnetic separations. The assay has a linear response in the 0.5 µg L-1 to 40 µg L-1 Clen concentration range, and 0.167 µg L-1 limit of detection. This makes it well suited for food safety monitoring. The average recoveries from spiked samples range from 92.7 to 109.1% (intra-assay) and 101.2 to 125.7% (inter-assay) with relative standard deviations of <11.6%. Spiked swine urine samples were analyzed by this method, and the results correlated well with data obtained by LC-MS/MS. Graphical abstract Fluorescent hybrid nanoflowers were fabricated with gold nanoclusters (BSA-AuNCs) and antigens. A fluorometric immunoassay based on the use of such nanoflowers and based on immunomagnetic separation was developed to detect clenbuterol residues in swine urine with satisfactory recoveries and acceptable accuracy.

Highly sensitive detection of clenbuterol in urine sample by using surface plasmon resonance immunosensor.

This study proposed the filtration method for removal of inhibitors from real urine samples for immunoassay without centrifuge. Although the inhibitors could not be removed by the physical filtration, the carboxyl group terminated silica effectively removed the inhibitors. In a low pH, antibody formed aggregation due to the protonation. We propose to adjust pH of the sample solution by adding a phosphate buffer solution (pH 7.5). As a result of pretreatment, the SPR immunosensing achieved the SPR signal of 45 mdeg and a low limit of detection with 100 ppq (100 fg mL-1).

Isolation and characterization of a novel anti-salbutamol chicken scFv for human doping urinalysis.

Anti-salbutamol antibodies remain as important tools for the detection of salbutamol abuse in athletic doping. This study evaluated the feasibility and efficiency of the chicken (Gallus gallus domesticus) as an immunization host to generate anti-salbutamol scFv antibodies by phage display. A phage display antibody library was constructed from a single chicken immunized against salbutamol-KLH conjugate. After a stringent biopanning strategy, a novel scFv clone which was inhibited by free salbutamol recorded the highest affinity. This scFv was expressed as soluble and functional protein in Escherichia coli T7 SHuffle Express B (DE3) strain. Cross-reactivity studies of the scFv towards other relevant ß2-agonists revealed that the scFv cross-reacted significantly towards clenbuterol. The determined IC50 of the scFv towards the two ß2-agonists were; IC50 salbutamol = ∼0.310 µg/ml, IC50 clenbuterol = ∼0.076 µg/ml. The generated scFv demonstrated poor stability based on accelerated stability studies. The scFv was used to develop an competitive indirect ELISA (LOD = 0.125 µg/ml) for detection of parent salbutamol in spiked human urine (n = 18) with ∼83.4% reliability at the cut-off of 1 µg/ml currently implemented by WADA and may be of potential use in human doping urinalysis.

Development of extremely stable dual functionalized gold nanoparticles for effective colorimetric detection of clenbuterol and ractopamine in human urine samples.

New glutamic acid (Glu) and polyethylenimine (PE) functionalized ultra-stable gold nanoparticles (PE-Glu-AuNPs) were developed via a simple NaBH4 reduction method. The low toxicity and biocompatibility of PE-Glu-AuNPs were confirmed via an MTT assay in Raw 264.7 cells. Excitingly, PE-Glu-AuNPs were found to be extremely stable at room temperature up to six months and were utilized in an effective colorimetric naked eye assay of clenbuterol (CLB) and ractopamine (RCT) at pH 5. It was found that the selective assay of CLB and RCT is not affected by any other interferences (such as alanine, phenylalanine, NaCl, CaCl2, threonine, cysteine, glycine, glucose, urea and salbutamol). Furthermore, the detection of these ß-agonists can be visually accomplished through change color from wine red to purple blue. Notably, the aggregation induced detection of CLB and RCT was well confirmed through transmission electron microscopy (TEM) and dynamic light scattering (DLS) studies. DLS investigations, clearly showed, that in the presence of CLB and RCT, the initial size of PE-Glu-AuNPs (12.8 ±â€¯8.6 nm) was changed to 84.8 ±â€¯52.3 and 79.5 ±â€¯47.8 nm, respectively, via aggregation. Furthermore, the colorimetric assays of CLB and RCT with PE-Glu-AuNPs were effective starting from CLB and RCT concentrations of 200 nM and 400 nM, respectively, and could be visualized using the naked eyes. Remarkably, UV-vis titrations of PE-Glu-AuNPs with CLB and RCT could be used to well estimate their sub nanomolar detection limits (LODs) via standard deviation and linear fittings. The contribution of surface functional groups that support the analyte recognition was confirmed by fourier-transform infrared spectroscopy (FTIR) analysis. Moreover, the CLB and RCT assays with PE-Glu-AuNPs were supported by examination of human urine samples.

Flow-batch analysis of clenbuterol based on analyte extraction on molecularly imprinted polymers coupled to an in-system chromogenic reaction. Application to human urine and milk substitute samples.

A fully automated spectrophotometric method based on flow-batch analysis has been developed for the determination of clenbuterol including an on-line solid phase extraction using a molecularly imprinted polymer (MIP) as the sorbent. The molecularly imprinted solid phase extraction (MISPE) procedure allowed analyte extraction from complex matrices at low concentration levels and with high selectivity towards the analyte. The MISPE procedure was performed using a commercial MIP cartridge that was introduced into a guard column holder and integrated in the analyzer system. Optimized parameters included the volume of the sample, the type and volume of the conditioning and washing solutions, and the type and volume of the eluent. Quantification of clenbuterol was carried out by spectrophotometry after in-system post-elution analyte derivatization based on azo-coupling using N- (1-Naphthyl) ethylenediamine as the coupling agent to yield a red-colored compound with maximum absorbance at 500nm. Both the chromogenic reaction and spectrophotometric detection were performed in a lab-made flow-batch mixing chamber that replaced the cuvette holder of the spectrophotometer. The calibration curve was linear in the 0.075-0.500mgL-1 range with a correlation coefficient of 0.998. The precision of the proposed method was evaluated in terms of the relative standard deviation obtaining 1.1% and 3.0% for intra-day precision and inter-day precision, respectively. The detection limit was 0.021mgL-1 and the sample throughput for the entire process was 3.4h-1. The proposed method was applied for the determination of CLB in human urine and milk substitute samples obtaining recoveries values within a range of 94.0-100.0%.

Nanodiamonds conjugated to gold nanoparticles for colorimetric detection of clenbuterol and chromium(III) in urine.

Nanodiamonds were modified such that they carry thiol groups (ND-thiol). Gold nanoparticles were reacted with ND-thiol to obtain a highly stable conjugate of the type ND@AuNPs. Both ND-thiol and the ND@AuNPs were characterized by SEM, TEM, AFM, DLS, zeta potential, XPS, XRD, UV-Vis, Raman, FTIR and cytotoxicity studies. Their biocompatibility was confirmed via an MTT assay with HeLa cells. At a pH value of 6, the ND@AuNPs represent a colorimetric probe that can be used to selectively detect the illegally used ß-adrenergic drug clenbuterol (CLB) and the pollutant chromium(III). Detection can be performed visually by monitoring the color change from wine red to purple blue, or by colorimetric measurement of the so-called SPR peaks at 651 and 710 nm. The color changes are due to aggregation, and this is confirmed by TEM and DLS data. The involvement of surface functional groups that assist in analyte recognition was verified by FTIR. The detection limits are 0.49 nM for CLB, and 0.37 nM for Cr(III). The ND@AuNPs were successfully applied to the determination of Cr(III) and CLB in spiked human urine samples. Notably, the low interference by other ions in the detection of Cr(III) in tap and lake water is confirmed by ICP-MS analyses. Graphical abstract Nanodiamonds carrying thiol groups (ND-Thiol) were conjugated to gold nanoparticles, and the resulting ND@AuNPs are shown to be viable probes for the colorimetric detection of sub-nanomolar levels of clenbuterol (CLB) and Cr(III) ions, with demonstrated applicability to real water and urine samples.

Molecularly Imprinted Membrane Electrospray Ionization for Direct Sample Analyses.

Typically dealing with practical samples with very complex matrices, ambient ionization mass spectrometry suffers from low detection sensitivity. In this study, molecular imprinting technology was explored and integrated with the membrane electrospray ionization (MESI) method for direct sample analyses. By enriching targeted analytes on molecularly imprinted membranes (MIMs), improvement (by 10- to 50-fold) in the limit of quantitation could be achieved, compared to conventional nanoelectrospray ionization methods or other ambient ionization methods. MIMs were prepared by cross-linking a synthesized molecularly imprinted polymer layer onto a polyvinylidene difluoride (PVDF) membrane. The characteristics of MIM in recognizing target analytes were investigated and verified. Experiments showed that MIM-ESI could provide satisfactory performances for direct quantification of targeted analytes in complex samples using mass spectroscopy (MS), and the quantitative performance of this methodology was validated. With the capability of target enrichment, the uses of MIM-ESI MS in different application fields were also demonstrated, including food safety, quantification of drug concentrations in blood, pesticide residues in soil, and antibiotic residues in milk.

Development of molecular imprinted column-on line-two dimensional liquid chromatography for selective determination of clenbuterol residues in biological samples.

A novel method coupling molecular imprinted monolithic column with two-dimensional liquid chromatography was developed and validated for the analysis of clenbuterol in pork liver and swine urine samples. The polymers were characterized by using Fourier transform infrared spectroscopy, nitrogen adsorption desorption analyses, frontal analysis and the adsorption of selectivity. The results indicated that the imprinted columns were well prepared and possessed high selectivity adsorption capacity. Subsequently, the MIMC-2D-LC (molecular imprinted monolithic column-two dimensional liquid chromatography) method was developed for the selective analysis of clenbuterol in practical samples. The accuracy ranged from 94.3% to 99.7% and from 93.7% to 99.6% for liver and urine, respectively. The relative standard deviation (RSD) of repeatability was lower than 8.6% for both analyses. The limit of detections was 16ng·mL(-1) for liver and 25ng·mL(-1) for urine, respectively. Compared with the reported methods, the disturbance of endogenous impurity could be avoided by the 2D-LC method.