Simultaneous determination of five beta-lactam antibiotics in bovine milk using liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A multiresidue method for the detection of five important beta-lactam antibiotics (amoxicillin, ampicillin, cloxacillin, oxacillin, penicillin G) in fresh milk is presented that allows quantitation of the analytes well below established legislative limits. The method avoids the use of acid during the extraction procedure and entails a cleanup step over a C18 cartridge. The analytes are separated and detected by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) using a stable isotope-labeled internal standard. Mass spectral acquisition is done in the positive ion mode by applying selected reaction monitoring of two or more fragmentation transitions per analyte to provide a high degree of sensitivity and specificity. The typical recoveries for all five beta-lactams in fresh milk ranged from 76 to 94% at a fortification level of 4 microg/kg. This study also addresses common problems encountered in the stability of penicillins during sample preparation as well as the employment of postcolumn infusion of a standard compound to verify potential matrix-induced signal suppression in ESI-MS.     

Sulfur-specific detection of impurities in cimetidine drug substance using liquid chromatography coupled to high resolution inductively coupled plasma mass spectrometry and electrospray mass spectrometry

The use of liquid chromatography coupled to sector field inductively coupled plasma mass spectrometry (SF-ICP-MS) for the specific detection of sulfur-containing compounds is described. In the sulfur-containing drug substance cimetidine, structurally related impurities well below the 0.1% mass fraction level relative to the main drug substance could easily be detected. The structure of most of the impurities was confirmed by electrospray mass spectrometry (ESI-MS), and thus, the complementarity of the two techniques for drug analysis is shown. The limit of detection by SF-ICP-MS for cimetidine in solution was approximately 4-20 ng x g(-1), but it was blank-limited.     

Protein phosphorylation degree: determination by capillary liquid chromatography and inductively coupled plasma mass spectrometry

Capillary liquid chromatography (muLC) interfaced to inductively coupled plasma mass spectrometry (ICPMS) is introduced as a new micromethod to determine the phosphorylation degree in phosphoproteins and phosphopeptides containing cysteine and/or methionine residues. The stoichiometric phosphorus to sulfur (31P to 32S) ratio is experimentally determined by muLC-ICPMS and converted into the degree of phosphorylation using protein/ peptide sequence information. The method is applied to the phosphoproteins beta-casein, beta-casein, and recombinant protein kinase A catalytic subunit and to synthetic phosphopeptides. The accurate data obtained by muLC-ICPMS allow quantitative assessment of the compound-specific discrimination of the electrospray ionization process between nonphosphorylated and phosphorylated proteins and peptides.     

Liquid chromatographic determination of cyclophosphamide enantiomers in plasma by precolumn chiral derivatization

On the basis of reactions described in the synthetic literature, a two-step chiral derivatization sequence was developed for the anticancer agent cyclophosphamide (CP). The sequence involves amidoalkylation of CP with anhydrous chloral containing 1% dimethylformamide followed by acylation of the resulting secondary alcohol with a chiral carboxylic acid chloride, (+)-6-methoxy-alpha-methyl-2-naphthaleneacetyl chloride, to form a diastereomeric pair. Derivatized (-)-CP and (+)-CP exhibited retention times of 17.2 and 20.7 min, respectively, when chromatographed on Hypersil ODS with acetonitrile/phosphate buffer (pH 6.8) as the mobile phase. Preparation of the individual diastereomers from enantiomerically pure CP enabled correlation of the chromatographically observed peaks with a particular enantiomer. Various aspects of the overall assay methodology have been systematically investigated (derivatization solvents, temperatures, reaction time, and work-up procedure) and optimized on the scale required for trace analysis in biological fluids. Calibration curves were established for each enantiomer in spiked human plasma over the therapeutically relevant concentration range of 0.99-49.94 micrograms/mL.     

Simultaneous determination of five tobacco-specific nitrosamines in mainstream cigarette smoke by isotope dilution liquid chromatography/electrospray ionization tandem mass spectrometry

Tobacco-specific nitrosamines (TSNAs) have been previously implicated as a source of carcinogenicity in tobacco and cigarette smoke. Accurate quantification of these chemicals is needed to help assess public health risk. We have developed and validated a specific and sensitive method to simultaneously measure five TSNAs in the particulate phase of mainstream tobacco smoke. Cigarette smoke particulate, produced using standardized machine smoking protocols, was collected on a Cambridge filter pad. The particulate matter was extracted using methylene chloride, back extracted into aqueous solution, further purified by solid-phase extraction, and analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry using isotopically labeled analogues as internal standards. Limits of detection for this method ranged from 0.05 to 1.23 ng/mL using an injection volume of 20 microL. A linear calibration range spanning 2.5-2500 ng/mL was adequate to measure TSNA levels in cigarette smoke. The method achieved excellent reproducibility and accuracy. The identity of each TSNA was established by chromatographic retention time, analyte-specific fragmentation patterns, and relative peak area ratios of two product/precursor ion pairs. This new method provides higher sensitivity, specificity, and throughput than earlier methods for TSNA determination.     

Analysis of protein phosphorylation by capillary liquid chromatography coupled to element mass spectrometry with 31P detection and to electrospray mass spectrometry

A method for phosphopeptide identification by capillary liquid chromatography (muLC) interfaced alternatively to element mass spectrometry (inductively coupled plasma mass spectrometry, ICPMS) and to electrospray ionization mass spectrometry (ESI-MS) is described. ICPMS is used for 31P detection and ESI-MS provides the corresponding molecular weight information. Alignment of the two separate muLC runs is performed using the baseline distortion at the elution front, which shows up in both muLC-ICPMS and muLC-ESI-MS. Both a quadrupole and a magnetic sector field mass analyzer were used in combination with ICP. The detection limit achieved for the muLC-ICP-HRMS runs is approximately 0.1 pmol of phosphopeptide injected. Without any further precautions, contamination by phosphate-containing compounds at this level was found to be uncritical. The method is demonstrated for the analysis of a complex mixture of synthetic phosphopeptides and a set of tryptic digests of three phosphoproteins. These include beta-casein, activated human MAP kinase ERK1, and protein kinase A catalytic subunit. The tryptic phosphopeptides of these proteins could all be detected and identified by our new strategy. Analysis of three fractions of protein kinase A catalytic subunit with different phosphorylation status gives direct access to the order in which the phosphorylation of the four phosphorylation sites occurs. The two most important aspects of using muLC-ICPMS with 31P detection for phosphopeptide identification are (i) that a high selectivity is achieved and (ii) that the signal intensity is independent of the chemical form of phosphorus and directly proportional to the molar amount of 31P in the muLC eluate. Thus, muLC-ICPMS with 31P detection is introduced as a new, robust, and specific method in phosphoproteomics.     

Simultaneous quantification of homocysteine and folate in human serum or plasma using liquid chromatography/tandem mass spectrometry

A unified extraction and quantification procedure based on stable isotope-dilution liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been developed for the simultaneous determination of total homocysteine and folate (5-methyltetrahydrofolic acid and folic acid) levels in human serum and plasma. This is the first report documenting the simultaneous extraction and quantification of these structurally dissimilar analytes. Analytes are quantitatively isolated from samples (500 microL) prior to LC/MS/MS analysis using a two-step stabilization process combined with C18 solid-phase extraction. The method exhibits excellent linearity over 4 orders of magnitude for each analyte. Measurement repeatability (RSD, N = 2) ranged from 0.3% to 3% for all analytes over 1 day of analysis. Total method variability (RSD, N = 6) ranged from 0.7% to 10% for all analytes over three independent days of analysis. The accuracy and practical applicability of the method were demonstrated by applying the method to the quantitative determination of each analyte in a new NIST serum Standard Reference Material (NIST SRM 1955 Homocysteine and Folate in Frozen Human Serum) and in a small subset of normal donor plasma samples.     

Hydrophilic interaction liquid chromatography coupled to electrospray mass spectrometry of small polar compounds in food analysis

Reversed-phase liquid chromatography (RPLC) is commonly used to analyze nonvolatile components in food. However, polar low-molecular-weight compounds such as hydrophilic amino acids, di- and tripeptides, and organic acids are often not sufficiently retained and represent a challenge for RPLC. Hydrophilic interaction liquid chromatography in combination with electrospray mass spectrometry (HILIC-ESI-MS) on a carbamoyl-derivatized stationary phase was successfully employed to separate free amino acids and small polar peptides in complex food matrixes such as wheat gluten hydrolysate and Parmesan cheese. Glutamyl dipeptides were separated in a sequence-specific order with peptides with N-terminal glutamic acid residues eluting prior to their reverse sequence analogues. ESI-MSn detection in the positive ionization mode provided the necessary information to unambiguously identify isobaric peptides due to their characteristic fragmentation patterns. The technique also proved useful to separate and identify glycoconjugates between amino acids and reducing sugars (Amadori compounds). The investigation of organic acids present in food used a mobile phase comprising ammonium acetate buffer at pH 7 and mass spectrometric detection in the negative ionization mode.     

Comparison of electrokinetics-based multidimensional separations coupled with electrospray ionization-tandem mass spectrometry for characterization of human salivary proteins

The foundation for saliva-based diagnostics is the development of a complete catalog of secreted proteins detectable in saliva. Besides protein complexity, the greatest bioanalytical challenge facing comprehensive analysis of saliva samples is related to the large variation of protein relative abundances including the presence of high-abundance proteins such as amylases, mucins, proline-rich proteins (PRPs), and secretory IgA complex. Among a number of electrokinetic separation techniques, transient capillary isotachophoresis/capillary zone electrophoresis (CITP/CZE) specifically targets trace amounts of proteins and thus reduces the range of relative protein abundances for providing unparallel advantages toward the identification of low-abundance proteins. By employing a CITP/CZE-based multidimensional separation platform coupled with electrospray ionization-tandem mass spectrometry (ESI-tandem MS), a total of 6112 fully tryptic peptides are sequenced at a 1% false discovery rate (FDR), leading to the identification of 1479 distinct human SwissProt protein entries. By comparing with capillary isoelectric focusing (CIEF) as another electrokinetics-based stacking approach, CITP/CZE not only offers a broad field of application but also is less prone to protein/peptide precipitation during the analysis. The ultrahigh resolving power of CITP/CZE is evidenced by the large number of distinct peptide identifications measured from each CITP fraction together with the low peptide fraction overlapping among identified peptides. Furthermore, when evaluating the protein sequence coverage by the number of distinct peptides mapping to each protein identification, the CITP-based proteome technology similarly achieves the superior performance with 674 proteins (46%) having three or more distinct peptides, 288 (19%) having two distinct peptides, and 517 (35%) having a single distinct peptide.     

Identification and quantitation of despropionyl-bezitramide in postmortem samples by liquid chromatography coupled to electrospray ionization tandem mass spectrometry.

A sensitive and specific method for the determination and quantitation of (despropionyl) bezitramide in postmortem samples using liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) is presented. The method is the result from a simple methodological transfer of a liquid chromatographic method with fluorescence detection (LC-FL) previously developed in our laboratory. A liquid-liquid back-extraction procedure using n-hexane isoamyl alcohol (93:7, v/v) as the extraction solvent is performed for a basic sample cleanup. N-Methyldespropionyl bezitramide is used as the internal standard. Chromatographic separation of the analytes of interest is achieved on a Hypersil ODS 5-micron column, using a 80:20 (v/v) mixture of 1.0 mM ammonium acetate and methanol/acetonitrile (50:50, v/v) and 1.0 mM ammonium acetate as the mobile phase. To obtain as high a sensitivity and selectivity as possible, a selected reaction-monitoring mass spectrometric technique is applied. In addition, low-energy collisional-activated dissociation (CAD) product ion spectra are recorded for a few samples. Calibration graphs are prepared for blood and urine, and good linearity is achieved over a concentration range of 1-150 ng/mL. The intra- and interassay coefficients of variation (CV%) for the analysis of quality control samples at 10 and 50 ng/mL concentration levels do not exceed 10.2% and percent of targets are within 12.1%. Postmortem samples (blood, urine, stomach contents, bile, liver, and kidney) from three fatalities, all suspected victims of drug overdoses, are analyzed, and the results are reported. The results obtained with LC-ESI-MS/MS are in close agreement with those obtained using the LC-FL method. Moreover, the isolates' identity and structure are confirmed by the CAD product ion spectra, thus allowing to make unequivocal conclusions about the prior intake of bezitramide by the three subjects.     

Combination of chiral capillary electrochromatography with electrospray ionization mass spectrometry: method development and assay of warfarin enantiomers in human plasma

The hyphenation of chiral capillary electrochromatography (CEC) with electrospray ionization mass spectrometry (ESI-MS) is very challenging but promising due to the fact that it combines sensitivity with high specificity and selectivity. In this work, CEC capillaries packed with (3R,4S)-Whelk-O1 chiral stationary phase were used for simultaneous enantioseparation of (+/-)-warfarin and its internal standard, (+/-)-coumachlor. Furthermore, both the chiral CEC separation and MS detection parameters were examined in detail. First, the influence of different column fabrication was investigated. Second, enantioseparation was optimized by varying CEC parameters, including acetonitrile concentration, buffer pH, and ionic strength. Under the optimum chiral CEC conditions, ESI-MS parameters such as sheath liquid pH and composition, sheath liquid flow rate, drying gas flow rate, drying gas temperature, nebulizer pressure, and fragmentor voltage were investigated to achieve maximum MS signals of the separated enantiomers. Finally, using solid-phase extraction as sample preparation method, (+/-)-warfarin spiked in 100-microL human plasma samples were analyzed. The calibration curves showed good linearity for both (R)-warfarin (R = 0.9979) and (S)-warfarin (R = 0.9978) enantiomers. The experimental limit of detection was approximately 25 ng/mL for both enantiomers. Even though the data are still preliminary, we can state with confidence that chiral CEC-ESI-MS has the potential to establish itself as a very powerful technique for the determination of enantiomeric ratios in human body fluid.     

Identification and quantification of flavonoids in human urine samples by column-switching liquid chromatography coupled to atmospheric pressure chemical ionization mass spectrometry

A rapid and sensitive high-performance liquid chromatographic mass spectrometric (HPLC-MS) method is described for the determination and quantification of 12 dietary flavonoid glycosides and aglycons in human urine samples. Chromatographic separation of the analytes of interest was achieved by column-switching, using the first column (a Zorbax 300SB C-3 column) for sample cleanup and eluting the heart-cut flavonoid fraction onto the second column (a Zorbax SB C-18 column) for separation and detection by ultraviolet and atmospheric pressure chemical ionization MS using single ion monitoring in negative mode. The fragmentor voltage was optimized with regard to maximum abundance of the molecular ion and qualifier ions of the analytes. Calibration graphs were prepared for urine, and good linearity was achieved over a dynamic range of 2.5-1000 ng/mL. The inter- and intraassay coefficients of variation for the analysis of the 12 different flavonoids in quality control urine samples were 12.3% on average (range 11.0-13.7%, n = 24, reproducibility) and the repeatability of the assay were 5.0% (mean, range 0.1-14.8%, n = 12). A subset of 10 urine samples from a human dietary intervention study with high and low flavonoid content was analyzed, and the results are reported.     

Trace determination of macrolide and sulfonamide antimicrobials, a human sulfonamide metabolite, and trimethoprim in wastewater using liquid chromatography coupled to electrospray tandem mass spectrometry

An analytical method has been developed and validated for the simultaneous trace determination of four macrolide antibiotics, six sulfonamides, the human metabolite N4-acetylsulfamethoxazole, and trimethoprim in wastewater. The method was validated for tertiary, secondary, and-unlike in previously published methods-also for primary effluents of municipal wastewater treatment plants. This wide range of application is necessary to thoroughly investigate the occurrence and fate of chemicals in wastewater treatment. Wastewater samples were enriched by solid-phase extraction, followed by reversed-phase liquid chromatography coupled to tandem mass spectrometry using positive electrospray ionization. Recoveries from all sample matrixes were generally above 80%, and the combined measurement uncertainty varied between 2 and 18%. Concentrations measured in tertiary effluents ranged between 10 ng/L for roxithromycin and 423 ng/L for sulfamethoxazole. Corresponding levels in primary effluents varied from 22 to 1450 ng/L, respectively. Trace amounts of these emerging contaminants reach ambient waters, since all analytes were not fully eliminated during conventional activated sludge treatment followed by sand filtration. In the case of sulfamethoxazole, the amount present as human metabolite N4-acetylsulfamethoxazole had to be taken into account in order to correctly assess the fate of sulfamethoxazole in wastewater treatment.     

Quantitative and wide-ranging profiling of phospholipids in human plasma by two-dimensional liquid chromatography/mass spectrometry

Normal-phase or reverse-phase liquid chromatography has been used in phospholipidomics for lipid separation prior to mass spectrometry analysis. However, separation using a single separation mode is often inadequate, as high-abundance phospholipids can mask large numbers of low-abundance lipids of interest. In order to detect and quantify low-abundance phospholipids, we present a novel two-dimensional (2D) approach for sensitive and quantitative global analysis of phospholipids. The methodology monitors individual glycerolipids and phospholipids through the use of a new quantitative normal-phase, solid-phase extraction procedure, followed by molecular characterization and relative quantification using an ion-trap Orbitrap equipped with a reverse-phase liquid chromatograph, with data processing by MS++ software. The CV (%) of the peak area of each lipid standard was less than 15% with this extraction method. When the method was applied to a liver sample, we could detect more phosphatidylserine (PS) compared to the previous method. Finally, our developed method was applied to Alzheimer's disease (AD) plasma samples. Several hundred peaks were detected from a 60 μL plasma sample. A partial-least-squares discriminant analysis (PLS-DA) plot using peak area ratio gave a unique group of PLS scores which could distinguish plasma samples of Alzheimer's disease (AD) patients from those of age-matched healthy controls.     

Characterization of the human cerebrospinal fluid phosphoproteome by titanium dioxide affinity chromatography and mass spectrometry

Biomarkers in the cerebrospinal fluid (CSF) may be important for the diagnosis of chronic degenerative disorders in the central nervous system including dementia. Existing CSF biomarkers for dementia, however, are relatively nonspecific. More specific markers may be found by targeting investigations based on knowledge of the molecular pathology of the disease in question. In Alzheimer's disease, hyperphosphorylation of the tau protein is a characteristic feature and thus a comprehensive characterization of the phosphoproteome of the CSF may be pursued to obtain a complete picture of phosphorylation aberrations in health and disease. Toward that goal we here describe a method for a comprehensive isolation and identification of phosphorylated tryptic peptides derived from CSF proteins using a simple sample preparation step and titanium dioxide-affinity chromatography followed by MALDI-TOF or LC-MS/MS linear ion-trap-FT mass spectrometry. Whereas not all previously reported phosphoproteins were found in normal CSF, we detected 56 putative novel phosphorylation sites in 38 proteins in addition to known sites. The approach seems to be a promising foundation for the discovery of new biomarkers embedded in the CSF phosphoproteome.     

Cobalt speciation study in the cobalt-cysteine system by electrospray ionization mass spectrometry and anion-exchange chromatography inductively coupled plasma atomic emission spectrometry

This paper describes the ability of the combination of electrospray ionization mass spectrometry (ESI-MS) and anion-exchange chromatography coupled with inductively coupled plasma atomic emission spectrometry (AEC-ICP-AES) for cobalt speciation study in the binary cobalt-cysteine system. ESI-MS, allowing the identification and the characterization of the analytes, is used as a technique complementary to AEC-ICP-AES, providing elemental information on the separated species. The methods have been developed through the study of samples containing Co2+ and 1-fold to 5-fold molar ratios of cysteine over a pH range 2.5 to 11. In each case, cobalt-cysteine complexes were characterized by ESI-MS in negative ion mode. AEC-ICP-AES allowed further separation and detection of the cobalt species previously characterized. The strong influence of pH and ligand-to-metal ratios on the nature and stoichiometry of the species is demonstrated. For the first time, a direct experimental speciation diagram of cobalt species has been established owing to these analytical techniques. This work is a promising basis for the speciation analysis of cobalt, since a good knowledge of cobalt speciation is of prime importance to better understanding its fate in biological and environmental media.     

Applications of Hadamard transform to gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry

Successful application of the Hadamard transform (HT) technique to gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) is described. Novel sample injection devices were developed to achieve multiple sample injections in both GC and LC instruments. Air pressure was controlled by an electromagnetic valve in GC, while a syringe pump and Tee connector were employed for the injection device in LC. Two well-known, abused drugs, 3,4-methylenedioxy-N-methylamphetamine (MDMA) and N, N-dimethyltryptamine (DMT), were employed as model samples. Both of the injection devices permitted precise successive injections, resulting in clearly modulated chromatograms encoded by Hadamard matrices. After inverse Hadamard transformation of the encoded chromatogram, the signal-to-noise (S/N) ratios of the signals were substantially improved compared with those expected from theoretical values. The S/N ratios were enhanced approximately 10-fold in HT-GC/MS and 6.8 in HT-LC/MS, using the matrices of 1023 and 511, respectively. The HT-GC/MS was successfully applied to the determination of MDMA in the urine sample of a suspect.     

液相色谱–串联质谱法同时测定烟草中的肌醇和奎尼酸

建立同时测定烟草中肌醇和奎尼酸的液相色谱–串联质谱(LC-MS/MS)方法。采用0.1 mol/L的乙酸铵水溶液对烟末进行超声提取后,经0.22μm水相滤膜过滤后直接进行LC-MS/MS分析。结果表明:肌醇在0.11～25 mg/g和奎尼酸在0.18～25 mg/g范围内,线性相关系数均≥0.999 0。肌醇的回收率在95.9%～98.6%之间,相对标准偏差(RSD)小于1.9%(n=6);奎尼酸的回收率在95.2%～97.4%之间,相对标准偏差(RSD)小于2.1%(n=6)。肌醇的方法检出限(LOD)为0.035 mg/g,定量限(LOQ)为0.11 mg/g;奎尼酸的方法检出限(LOD)为0.055 mg/g,定量限(LOQ)为0.18 mg/g。该方法准确、灵敏,适合用于烟草中肌醇和奎尼酸的测定。     

Application of on-line electrochemical derivatization coupled with high-performance liquid chromatography electrospray ionization mass spectrometry for detection and quantitation of (p-chlorophenyl)aniline in biological samples

A rapid, sensitive, and specific assay for detection and quantitation of (p-chlorophenyl)aniline (CPA) in biological samples was developed. The assay was established based on rapid electrochemical oxidation of CPA to a dimerized product (1.0 V vs Pd) with the enhanced detection sensitivity of electrospray mass spectrometer (ES/MS). A "head-to-tail" dimer ([M + H]+ at m/z 217) was exhibited as the predominant species after electrochemical conversion of CPA. Optimal detection sensitivity and specificity for the dimer of CPA that was present in the biological matrix (e.g., rat urine) were achieved through on-line electrochemistry (EC) coupled with high-performance liquid chromatography tandem mass spectrometry. No matrix-associated ion suppression was observed. The limit of detection (S/N approximately 6) was 20 ng/mL, and the limit of quantitation was 50 ng/mL. The calibration curve was exhibited to be quadratic over the range of 50-2000 ng/mL with r2 > 0.99 in various biological matrixes. The assay was validated and used to study the biotransformation of p-chlorophenyl isocyanate (CPIC) to CPA in rats administered intraperitoneally with CPIC (50 mg/kg). The present LC/EC/MS/MS assay of CPA brings important technical advantages to assist in the risk assessment of new chemical entities, which have the potential to produce anilines via biotransformation.     

Automated in-tube solid-phase microextraction coupled with liquid chromatography/electrospray ionization mass spectrometry for the determination of beta-blockers and metabolites in urine and serum samples.

The technique of automated in-tube solid-phase microextraction (SPME) coupled with liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) was evaluated for the determination of beta-blockers in urine and serum samples. In-tube SPME is an extraction technique for organic compounds in aqueous samples, in which analytes are extracted from the sample directly into an open tubular capillary by repeated draw/eject cycles of sample solution. LC/MS analyses of beta-blockers were initially performed by liquid injection onto a LC column. Nine beta-blockers tested in this study gave very simple ESI mass spectra, and strong signals corresponding to [M + H]+ were observed for all beta-blockers. The beta-blockers were separated with a Hypersil BDS C18 column using acetonitrile/methanol/water/acetic acid (15:15:70:1) as a mobile phase. To optimize the extraction of beta-blockers, several in-tube SPME parameters were examined. The optimum extraction conditions were 15 draw/eject cycles of 30 microL of sample in 100 mM Tris-HCl (pH 8.5) at a flow rate of 100 microL/min using an Omegawax 250 capillary (Supelco, Bellefonte, PA). The beta-blockers extracted by the capillary were easily desorbed by mobile-phase flow, and carryover of beta-blockers was not observed. Using in-tube SPME/LC/ESI-MS with selected ion monitoring, the calibration curves of beta-blockers were linear in the range from 2 to 100 ng/mL with correlation coefficients above 0.9982 (n = 18) and detection limits (S/N = 3) of 0.1-1.2 ng/mL. This method was successfully applied to the analysis of biological samples without interference peaks. The recoveries of beta-blockers spiked into human urine and serum samples were above 84 and 71%, respectively. A serum sample from a patient administrated propranolol was analyzed using this method and both propranolol and its metabolites were detected.