Liquid Chromatography-tandem Mass Spectrometry Detection Research Articles

Detection of testosterone esters in blood.

Injections of synthetic esters of testosterone are among the most common forms of testosterone application. In doping control, the detection of an intact ester of testosterone in blood gives unequivocal proof of the administration of exogenous testosterone. The aim of the current project was to investigate the detection window for injected testosterone esters as a mixed substance preparation and as a single substance preparation in serum and plasma. Furthermore, the suitability of different types of blood collection devices was evaluated. Collection tubes with stabilizing additives, as well as non-stabilized serum separation tubes, were tested. A clinical study with six participants was carried out, comprising a single intramuscular injection of either 1000 mg testosterone undecanoate (Nebido(®)) or a mixture of 30 mg testosterone propionate, 60 mg testosterone phenylpropionate, 60 mg testosterone isocaproate, and 100 mg testosterone decanoate (Sustanon(®)). Blood was collected throughout a testing period of 60 days. The applied analytical method for blood analysis included liquid-liquid extraction and preparation of oxime derivatives, prior to TLX-sample clean-up and liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection. All investigated testosterone esters could be detected in post-administration blood samples. The detection time depended on the type of ester administered. Furthermore, results from the study show that measured blood concentrations of especially short-chained testosterone esters are influenced by the type of blood collection device applied. The testosterone ester detection window, however, was comparable.

Use of ‘dilute-and-shoot’ liquid chromatography-high resolution mass spectrometry in preclinical research: application to a DMPK study of perhexiline in mouse plasma

This work describes a simple, sensitive and rapid liquid chromatography–high resolution mass spectrometry method for the quantitation of perhexiline and the simultaneous detection of perhexiline metabolites in C57bl/6 mice plasma.Only 5μL of plasma was used for analysis. Pretreatment was limited to a 100-fold dilution (‘dilute-and-shoot’). The analyte was detected by high resolution mass spectrometry (Orbitrap™ technology). Three scan events were performed over the entire chromatogram. Targeted single ion monitoring with data dependent acquisition was employed for perhexiline quantitation and confirmation, while full scan was used to perform untargeted detection of perhexiline phase I and phase II circulating metabolites. The calibration curve was linear (r2=0.990) ranging from 0.305ng/mL (LLOQ) to 10000ng/mL. Matrix effect was limited to 6.1%.The method was applied to a pharmacokinetic study of perhexiline in mouse plasma and the results obtained were compared to a standard sample preparation method based on protein precipitation and liquid chromatography-tandem mass spectrometry (MRM mode) detection. The new approach provided comparable results in terms of pharmacokinetics parameters estimate with a high sensitivity, additional information on perhexiline circulating metabolites and a low consumption of biological sample.The combination of the ‘dilute-and-shoot’ approach together with HRMS targeted and untargeted detection represents a suitable alternative to classic bioanalytical approaches in preclinical research.

Carotenoid profile of tomato sauces: effect of cooking time and content of extra virgin olive oil.

The consumption of carotenoid-rich vegetables such as tomatoes and tomato sauces is associated with reduced risk of several chronic diseases. The predominant carotenoids in tomato products are in the (all-E) configuration, but (Z) isomers can be formed during thermal processing. The effect of cooking time (15, 30, 45 and 60 min) and the addition of extra virgin olive oil (5% and 10%) on the carotenoid extractability of tomato sauces was monitored using liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) and LC-ultraviolet detection (LC-UV). The thermal treatment and the addition of extra virgin olive oil increased the levels of antioxidant activity, total carotenoids, Z-lycopene isomers, α-carotene and β-carotene. These results are of particular nutritional benefit since higher lycopene intake has been associated with a reduced risk of lethal prostate and a reduction of prostate-specific antigen (PSA) levels. Moreover, β-carotene has been reported to suppress the up-regulation of heme oxygenase-1 gene expression in a dose dependent manner and to suppress UVA-induced HO-1 gene expression in cultured FEK4.

Coadministration of HMG-CoA reductase inhibitors, atorvastatin and rosuvastatin, does not affect contraceptive efficacy of centchroman

Objective The study aimed to investigate the effect of concomitant use of atorvastatin or rosuvastatin on the pharmacokinetic and pharmacodynamic profile of centchroman, a non-steroidal female oral contraceptive.Methods A rat model was used to predict pharmacokinetic drug–drug interactions between centchroman and atorvastatin or rosuvastatin. A dried blood spot sampling technique followed by liquid chromatography–tandem mass spectrometry detection was employed for analysis of the pharmacokinetic interaction study samples. Sperm-positive female rats were investigated for postcoital contraceptive activity of centchroman with or without coadministration of atorvastatin or rosuvastatin.Results Coadministration of atorvastatin or rosuvastatin may increase the systemic availability of centchroman in blood, but it does not affect the pharmacodynamic profile of centchroman.Conclusion Atorvastatin or rosuvastatin may be prescribed with centchroman without compromising the contraceptive efficacy of centchroman.Chinese 摘要目的 这项研究旨在调查同时使用阿托伐他汀或罗苏伐他汀时centchroman（一种非甾体女性口服避孕药）的药代动力学和药效情况。方法 采用大鼠模型来预测centchroman与阿托伐他汀或罗苏伐他汀之间药物相互作用的药代动力学。随后采用干燥血斑点采样技术和液相色谱串联质谱检测用于样本的药代动力学相互作用的研究分析。对精子阳性的雌性大鼠，观察centchroman同时服用/不服用阿托伐他汀或瑞舒伐他汀的事后避孕效果。结果 合用阿托伐他汀或瑞舒伐他汀可能会增加全身血液中的centchroman的生物利用率，但并不影响centchroman的药效。结论 阿托伐他汀或瑞舒伐他汀并不影响centchroman的避孕效果

Dissipation and Residue Level of Thifluzamide in Rice Field Ecosystem

An efficient modified QuEChERS method combined with high performance liquid chromatography-tandem mass spectrometry detection (HPLC-MS/MS) was established and evaluated for the residue analysis of thifluzamide in rice grain, husk, straw, seedling, paddy water, and soil. Thifluzamide residues were extracted with acetonitrile, cleaned up with primary secondary amine (PSA), and then determined by HPLC-MS/MS. The fortified recoveries were 76%–106% with RSDs of 3%–13%. The results of the supervised field trials at two experiment sites showed that thifluzamide dissipated rapidly in paddy fields, and the half-lives in paddy water, soil, and rice seedling were 0.3–0.6 d, 1.8–3.6 d, and 4.3–13.9 d, respectively. At harvest time, when the preharvest interval (PHI) was set as 21 d, the final residues of thifluzamide in rice grains were below the maximum residue limit (MRL) of 0.5 mg/kg set by Japan, whereas the final residues in rice husk and straw were still high (the highest value reached 1.36 mg/kg in rice husk and 0.83 mg/kg in rice straw). The results indicated that the highest residue in rice grain was 0.23 mg/kg when PHI was 21 d, and only 6.9–11.0% of acute risk quotient of thifluzamide was occupied by the dietary daily intake in Chinese population consuming rice.

Investigation of the Persistence of Nerve Agent Degradation Analytes on Surfaces through Wipe Sampling and Detection with Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectrometry

The persistence of chemical warfare nerve agent degradation analytes on surfaces is important, from indicating the presence of nerve agent on a surface to guiding environmental restoration of a site after a release. Persistence was investigated for several chemical warfare nerve agent degradation analytes on indoor surfaces and presents an approach for wipe sampling of surfaces, followed by wipe extraction and liquid chromatography-tandem mass spectrometry detection. Commercially available wipe materials were investigated to determine optimal wipe recoveries. Tested surfaces included porous/permeable (vinyl tile, painted drywall, and wood) and largely nonporous/impermeable (laminate, galvanized steel, and glass) surfaces. Wipe extracts were analyzed by ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). UPLC provides a separation of targeted degradation analytes in addition to being nearly four times faster than high-performance liquid chromatography, allowing for greater throughput after a large-scale contamination incident and subsequent remediation events. Percent recoveries from nonporous/impermeable surfaces were 60-103% for isopropyl methylphosphonate (IMPA), GB degradate; 61-91% for ethyl methylphosphonate (EMPA), VX degradate; and 60-98% for pinacolyl methylphosphonate (PMPA), GD degradate. Recovery efficiencies for methyl phosphonate (MPA), nerve agent degradate, and ethylhydrogen dimethylphosphonate (EHDMAP), GA degradate, were lower, perhaps due to matrix effects. Diisopropyl methylphosphonate, GB impurity, was not recovered from surfaces. The resulting detection limits for wipe extracts were 0.065 ng/cm(2) for IMPA, 0.079 ng/cm(2) for MPA, 0.040 ng/cm(2) for EMPA, 0.078 ng/cm(2) for EHDMAP, and 0.013 ng/cm(2) for PMPA. The data indicate that laboratories may hold wipe samples for up to 30 days prior to analysis. Target analytes were observed to persist on surfaces for at least 6 weeks.

Derivatization of the tricarboxylic acid intermediates with O-benzylhydroxylamine for liquid chromatography–tandem mass spectrometry detection

The tricarboxylic acid (TCA) cycle is an interface among glycolysis, lipid metabolism, and amino acid metabolism. Increasing interest in cancer metabolism has created a demand for rapid and sensitive methods for quantifying the TCA cycle intermediates and related organic acids. We have developed a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method to quantify the TCA cycle intermediates in a 96-well format after O-benzylhydroxylamine (O-BHA) derivatization under aqueous conditions. This method was validated for quantitation of all common TCA cycle intermediates with good sensitivity, including α-ketoglutarate, malate, fumarate, succinate, 2-hydroxyglutarate, citrate, oxaloacetate, pyruvate, isocitrate, and lactate using a 8-min run time in cancer cells and tissues. The method was used to detect and quantify changes in metabolite levels in cancer cells and tumor tissues treated with a pharmacological inhibitor of nicotinamide phosphoribosyl transferase (NAMPT). This method is rapid, sensitive, and reproducible, and it can be used to assess metabolic changes in cancer cells and tumor samples.

Development of a sensitive method for the quantification of urinary 3-hydroxybenzo[a]pyrene by solid phase extraction, dansyl chloride derivatization and liquid chromatography-tandem mass spectrometry detection

A sensitive method for the quantification of urinary 3-OHBaP was developed by SPE, dansyl chloride derivatization and LC-MS/MS detection.

Development of “one-pot” method for multi-class compounds in porcine formula feed by multi-function impurity adsorption cleaning followed ultra-performance liquid chromatography–tandem mass spectrometry detection

A novel and efficient determination method for multi-class compounds including β-agonists, sedatives, nitro-imidazoles and aflatoxins in porcine formula feed based on a fast "one-pot" extraction/multifunction impurity adsorption (MFIA) clean-up procedure has been developed. 23 target analytes belonging to four different class compounds could be determined simultaneously in a single run. Conditions for "one-pot" extraction were studied in detail. Under the optimized conditions, the multi-class compounds in porcine formula feed samples were extracted and purified with methanol contained ammonia and absorbents by one step. The compounds in extracts were purified by using multi types of absorbent based on MFIA in one pot. The multi-walled carbon nanotubes were employed to improved clean-up efficiency. Shield BEH C18 column was used to separate 23 target analytes, followed by tandem mass spectrometry (MS/MS) detection using an electro-spray ionization source in positive mode. Recovery studies were done at three fortification levels. Overall average recoveries of target compounds in porcine formula feed at each levels were >51.6% based on matrix fortified calibration with coefficients of variation from 2.7% to 13.2% (n=6). The limit of determination (LOD) of these compounds in porcine formula feed sample matrix was <5.0 μg/kg. This method was successfully applied in screening and confirmation of target drugs in >30 porcine formula feed samples. It was demonstrated that the integration of the MFIA protocol with the MS/MS instrument could serve as a valuable strategy for rapid screening and reliable confirmatory analysis of multi-class compounds in real samples.

Fate of enniatins and beauvericin during the malting and brewing process determined by stable isotope dilution assays

The fate of enniatins A, A1, B, B1 and beauvericin during the malting and brewing process was investigated. Three batches of barley grains were used as starting material, one was naturally contaminated, two were artificially inoculated with Fusarium fungi. Samples were taken from each key step of the malting and brewing procedure, the levels of the toxins were determined with stable isotope dilution assays using liquid chromatography–tandem mass spectrometry detection. Significant increases of the toxins were found during germination of two batches of barley grains, resulting in green malts contamination up to a factor of 3.5 compared to grains before germination. Quantitative PCR analyses of fungal DNA revealed in all batches growth of Fusarium avenaceum during germination. After kilning, only 41–72% of the total amounts of the toxins in green malts remained in kilned malts. In subsequent mashing stage, the toxins in kilned malts predominantly were removed with spent grains. In the final beer, only one batch still contained 74 and 14 μg/kg of enniatin B and B1, respectively. Therefore, the carryover of these enniatins from the initial barley to final beer was less than 0.2% with the main amounts remaining in the spent grains and the malt rootlets.

Investigation of different sample pre-treatment routes for liquid chromatography–tandem mass spectrometry detection of caseins and ovalbumin in fortified red wine

Different sample treatment protocols for the liquid chromatography–electrospray-tandem mass spectrometry (LC–ESI-MS/MS) analysis of potential residuals of ovalbumin and caseins added to red wines were developed. In particular, attention was paid to the simultaneous detection and quantitation of fining agent residues, i.e. ovalbumin, α- and β-casein, in wine samples. The different sample treatment methods were compared in terms of protein recovery. The use of denaturing agents combined with size exclusion concentration and purification allowed to obtain a reproducible (RDS < 20%) analytical protocol with good recoveries (73(±2) – 109(±4)% range) for digested proteins from 12.5 mL of wine sample. Matrix-matched calibration from LC–ESI-MS/MS analysis indicated that the devised method allowed detection of target peptides in the 0.01–0.8 μg/mL range. Finally, method applicability and selectivity was demonstrated by using fining agents commonly exploited in winery industry and by analyzing 20 commercial red wine samples.

Hydrophilic Material for the Selective Enrichment of 5-Hydroxymethylcytosine and Its Liquid Chromatography–Tandem Mass Spectrometry Detection

5-Methylcytosine (5-mC), an important epigenetic modification involved in development, can be converted enzymatically to 5-hydroxymethylcytosine (5-hmC). 5-hmC is considered an intermediate of active DNA cytosine demethylation and makes itself serve as an epigenetic mark. 5-hmC content in most mammalian cells is low and the quantification of 5-hmC by liquid chromatography-mass spectrometry (LC-MS) frequently suffers from ion suppression by the presence of unmodified nucleosides. To circumvent this problem, we developed a method to selectively transfer a glucosyl group to the hydroxymethyl moiety of 5-hmC and form a more hydrophilic residue (β-glucosyl-5-hydroxymethyl-2'-deoxycytidine, 5-gmdC) by using T4 β-glucosyltransferase. The more hydrophilic 5-gmdC can be selectively enriched by using NH2-silica via hydrophilic interaction prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, which eliminates the ion suppression and significantly improves the detection sensitivity and accuracy. Using this method, we successfully quantified 5-hmC content in genomic DNA of three human cell lines and seven yeast strains. To the best of our knowledge, this is the first report about the existence of 5-hmC in the model organism of yeast. In addition, the contents of 5-hmC in two yeast strains of Schizosaccharomyces pombe are even higher than those of 5-mC, indicating that 5-hmC may play important roles on the physiological functions of yeast.

Quantification of Isotope-Labeled and Unlabeled Folates and Folate Catabolites in Urine Samples by Stable Isotope Dilution Assay

Dual-label stable isotope dilution assays for the simultaneous quantification of isotopologic folates in clinical samples offer the perspective for differentiating between unlabeled folates from endogenous body pools and administered [13C5]-labeled folates from a test dose when performing bioavailability trials. In contrast to intact folates, this methodology could hitherto not be applied to the quantification of the folate catabolites, p-aminobenzoyl glutamate and p-acetamidobenzoyl glutamate. In this study, [2H4]-p-aminobenzoyl glutamate, [2H4]-p-acetamidobenzoyl glutamate, and unlabeled p-acetamidobenzoyl glutamate were synthesized. The synthesis of the [2H4]-labeled compounds started at unlabeled p-aminobenzoic acid. For the formation of p-acetamidobenzoyl glutamate, p-aminobenzoyl glutamate was acetylated. The new substances were applied successfully in stable isotope dilution assays for the simultaneous quantification of the [13C5]-labeled and unlabeled folate catabolites, p-aminobenzoyl glutamate and p-acetamidobenzoyl glutamate, along with the predominant folate vitamers in urine. The assays were based on clean-up by strong anion exchange followed by liquid chromatography-tandem mass spectrometry detection. Assay sensitivity was sufficient to detect the folate catabolites in physiologic concentrations. The limit of detection was below 0.4 and 0.3 nmol/100 g for p-aminobenzoyl glutamate isotopologues and p-acetamidobenzoyl glutamate isotopologues in urine, respectively. The successful synthesis of [2H4]-p-aminobenzoyl glutamate, [2H4]-p-acetamidobenzoyl glutamate, and unlabeled p-acetamidobenzoyl glutamate and the implementation of these substances in stable isotope dilution assays allows dual-label designs that provide a more detailed insight into human folate metabolism.

Determination of β2-Agonists in Porcine Urine by Molecularly Imprinted Solid Phase Extraction Followed Ultra-Performance Liquid Chromatography–Tandem Mass Spectrometry Detection

A novel, sensitive, and robust method has been developed to detect 9 β2-agonists in porcine urine to monitor illegal use of β2-agonists in swine rearing. The method based on the molecular imprinted polymer (MIP) rapid extraction followed ultra-performance liquid chromatography coupled tandem mass spectrometry (UPLC-MS/MS) detection. The cleaning efficiency of MIP cartridges was demonstrated by comparing with common ion exchange solid phase extraction. The presented method was validated in accordance with the European Commission Decision 2002/657/EC. The linearity, decision limit (CCα), detection capability (CCβ), recovery, precision, robustness, and stability were studied in detail. CCα and CCβ values were from 0.006 ng/mL to 0.03 ng/mL and from 0.02 ng/mL to 0.08 ng/mL, respectively. The mean recoveries and repeatability varied from 68.8% to 94.2% and from 2.8% to 10.1%. The proposed method was applied to test 170 porcine urine samples from the Shaanxi province in China and two urine samples were confirmed as clenbuterol positive and the concentrations of clenbuterol in positive urine samples were about 0.08 ng/mL and 0.1 ng/mL, respectively. The developed method was demonstrated to be more sensitive and robust for the determination of 9 β2-agonists in porcine urine. The method was proven to be simple and easy in operation with high selectivity and good reproducibility.

Wipe selection for the analysis of surface materials containing chemical warfare agent nitrogen mustard degradation products by ultra-high pressure liquid chromatography–tandem mass spectrometry

Degradation products arising from nitrogen mustard chemical warfare agent were deposited on common urban surfaces and determined via surface wiping, wipe extraction, and liquid chromatography–tandem mass spectrometry detection. Wipes investigated included cotton gauze, glass fiber filter, non-woven polyester fiber and filter paper, and surfaces included several porous (vinyl tile, painted drywall, wood) and mostly non-porous (laminate, galvanized steel, glass) surfaces. Wipe extracts were analyzed by ultra-high pressure liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) and compared with high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) results. An evaluation of both techniques suggests UPLC–MS/MS provides a quick and sensitive analysis of targeted degradation products in addition to being nearly four times faster than a single HPLC run, allowing for greater throughput during a wide-spread release concerning large-scale contamination and subsequent remediation events. Based on the overall performance of all tested wipes, filter paper wipes were selected over other wipes because they did not contain interferences or native species (TEA and DEA) associated with the target analytes, resulting in high percent recoveries and low background levels during sample analysis. Other wipes, including cotton gauze, would require a pre-cleaning step due to the presence of large quantities of native species or interferences of the targeted analytes. Percent recoveries obtained from a laminate surface were 47–99% for all nitrogen mustard degradation products. The resulting detection limits achieved from wipes were 0.2ng/cm2 for triethanolamine (TEA), 0.03ng/cm2 for N-ethyldiethanolamine (EDEA), 0.1ng/cm2 for N-methyldiethanolamine (MDEA), and 0.1ng/cm2 for diethanolamine (DEA).

Analysis of the Polymer from Extracts in Oil Shale

One kind of polymer with m/z 113 was found from extracts in oil shale using liquid chromatography tandem mass spectrometry (LC-MS). The polymer studied in this report was as follows: a preparation and purification method of extraction was set up, a LC-MS detection measure of the polymer was established and the splitting way of the polymer was verified rationally.

Determination of avermectin and milbemycin residues in bovine muscle by liquid chromatography-tandem mass spectrometry and fluorescence detection using solvent extraction and low temperature cleanup

A simple and inexpensive sample preparation method based on solvent extraction, followed by low temperature cleanup, was demonstrated to be applicable for the determination of avermectin and milbemycin residues in bovine muscle by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography with fluorescence (LC-FL) detection. The analytical methodology was validated according to the Commission Decision 2002/657/EC, using LC-MS/MS for confirmatory and LC-FL for quantitative purposes. Mean recovery was between 88.9 and 100.7% in three distinct concentrations. The coefficient of variation for repeatability and within-laboratory reproducibility ranged from 0.78 to 5.1% and from 0.28 to 9.0%, respectively. Method precision led to satisfactory values of decision limits (CCα) and detection capabilities (CCβ). The proposed method has been applied in the Brazilian National Residue Control Plan since 2010 for the determination of avermectins and milbemycin residues in bovine muscle samples. A total of 760 samples were analyzed and none of them presented residues at concentrations above the permitted levels established by the more recently applied directives.

CONFIRMATION OF THE ABSENCE OF BISPHENOL A IN COPOLYESTERS BY POLYMER HYDROLYSIS IN COMBINATION WITH LC-MS/MS AND LC-FLD

A method was developed to validate that certain polyester and copolyester resins do not contain bisphenol A (BPA). This method satisfies the need for a timely screening method that can be utilized on a timescale that is appropriate for a laboratory in a manufacturing facility. This was accomplished by hydrolyzing the polymer and then analyzing the hydrolysate by either liquid chromatography-tandem mass spectrometry (LC-MS/MS) or liquid chromatography-fluorescence detection (LC-FLD). A hydrolysis procedure offers the unique advantage of a faster sample preparation step as compared to extraction procedures. The hydrolysis was complete after 20 min and the procedure was fully automated with the use of an autosampler. By hydrolyzing the polymer, total BPA is measured rather than only extractable monomeric BPA. Copolyester samples were purposely spiked with polycarbonate to validate the method. The detection limit for the LC-MS/MS method was 0.8 mg kg−1 in the polymer, while the detection limit for the LC-FLD method was 2.6 mg kg−1 in the polymer.

Rapid determination of yunaconitine and related alkaloids in aconites and aconite‐containing drugs by ultra high‐performance liquid chromatography–tandem mass spectrometry

Yunaconitine (YAC) is a toxic aconite alkaloid that is considered to be a hidden aconite poison since it is frequently found in body fluids from aconite poisoning patients, but has not been well studied in commonly used herbal drugs. In this paper, a rapid and sensitive ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) detection combined with microwave-assisted extraction (MAE) was developed for high throughput simultaneous determination of YAC and six other toxic aconite alkaloids in 31 samples of crude, processed aconites and aconite-containing drugs. The optimized method showed excellent linearity, precision, accuracy and recovery for all target compounds with short run time. YAC was detected in some samples with contents from 0.015 to 10.41 mg/g. This is the first report on the determination of YAC in Radix Aconiti, Radix Aconiti Kusnezoffii and aconite-containing drugs. This newly developed method facilitates the rapid screening of YAC and related toxic aconite alkaloids and allows YAC to be used as a chemical marker for the quality control of aconites and aconite-containing drugs.

Validation of an on-line solid-phase extraction method coupled to liquid chromatography–tandem mass spectrometry detection for the determination of Indacaterol in human serum

Indacaterol has been recently approved in Europe for the treatment of chronic obstructive pulmonary disease (COPD). In the present study, we have developed and validated a rapid and sensitive on-line solid phase extraction (SPE) method coupled to liquid chromatography–tandem mass spectrometry (LC–MS/MS) detection for the determination of Indacaterol in human serum. The sample preparation involves the serum dilution with a 0.2% acetic acid solution prior to the on-line SPE on a mixed-mode cationic (MCX) polymer based sorbent. The samples were then eluted on a reversed phase column with a mobile phase made of acidified water and methanol and detection was performed by MS using electrospay ionization in positive mode. The analysis time between 2 samples was 7.0min. Standard curves were linear over the range of 10.0pg/mL (LLOQ) to 1000pg/mL with correlation coefficient (r2) greater than 0.990. The method specificity was demonstrated in six different batches of human serum. Intra-run and inter-run precision and accuracy within ±20% (at the LLOQ) and ±15% (other levels) were achieved during a 3-run validation for quality control samples (QCs). The stability at room temperature (38h) was determined and reported. In addition, the comparison between an off-line SPE procedure and our method gave equivalent results. The results of the present work demonstrated that our on-line SPE–LC–MS/MS method is rapid, sensitive, specific and could be applied to the quantitative analysis of Indacaterol in human serum samples. Our method effectively eliminated the tedious conditioning and rinsing steps associated with conventional off-line SPE and reduced the analysis time. The on-line SPE approach appears attractive for supporting the analysis of several hundreds of clinical samples.