In-house validation of a liquid chromatography/electrospray tandem mass spectrometry method for confirmation of chloramphenicol residues in muscle according to Decision 2002/657/EC

In this work we present an in-house validation study for the confirmatory analysis of chloramphenicol (CAP) in muscle according to the Commission Decision 2002/657/EC requirements. CAP is extracted in acetonitrile and after liquid-liquid partitioning with n-hexane is identified and quantitatively determined by ion trap liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) analysis in the negative ion mode. CAP was identified using the precursor ion and at least two product ions, meeting the qualitative and quantitative criteria set by the European Commission in the Decision 2002/657/EC for confirmation of prohibited veterinary drug residues. We calculated mean drug recoveries, CCalpha and CCbeta of the method, and reported data on specificity, ruggedness and within-laboratory reproducibility. Finally, we point out and discuss some problems and questions arising from controversy about the application of Decision 2002/657/EC.     

Tetracycline residues in royal jelly and honey by liquid chromatography tandem mass spectrometry: validation study according to Commission Decision 2002/657/EC

A specific, sensitive and robust liquid chromatography tandem mass spectrometry method for determining oxytetracycline, tetracycline, chlortetracycline and doxycycline in royal jelly and honey samples is presented. Extraction of drug residues was performed by ammonium acetate buffer as extractant followed by a clean-up with metal chelate affinity chromatography and solid-phase extraction. Tetracycline analysis was performed using liquid chromatography–electrospray ionisation–tandem mass spectrometry. The presented method is the first validated for royal jelly and in accordance with the requirements set by Commission Decision 2002/657/EC. Recoveries of the methods, calculated spiking the samples at 5.0, 10.0, 20.0 and 30.0 μg kg⁻¹, were 79% to 90% for honey and 77% to 90% for royal jelly. The intra-day precision (RSD) ranged between 8.1% and 15.0% for honey and from 9.1% to 16.3% for royal jelly, while inter-day precision values were from 10.2% to 17.6% and from 10.6% to 18.4% respectively for honey and royal jelly. Linearity for the four analytes was calculated from 5.0 to 50.0 μg kg⁻¹. The decision limits (CCα) ranged from 6.2 to 6.4 μg kg⁻¹ and from 6.1 to 6.5 μg kg⁻¹ for honey and royal jelly, respectively. Detection capabilities values (CCβ) ranged between 7.2 and 7.7 μg kg⁻¹ and from 7.3 to 7.9 μg kg⁻¹ respectively for honey and royal jelly. The developed method is currently in use for confirmation of the official control analysis of honey and royal jelly samples.     

Use of capillary electrophoresis with laser-induced fluorescence detection to screen and liquid chromatography–tandem mass spectrometry to confirm sulfonamide residues: Validation according to European Union 2002/657/EC

A multiresidue method is described for determining six sulfonamides (SAs) (sulfadiazine, sulfathiazole, sulfamethazine, sulfamethoxazole, sulfaquinoxaline and sulfadimethoxine) in liver by a capillary electrophoresis screening method and a liquid chromatography coupled to tandem mass spectrometry confirmatory assay. Samples were prepared by homogenizing the tissue, with sodium hydroxide and acetonitrile. After evaporation, extracts were injected in the capillary electrophoresis system or mass spectrometry system for confirmatory analysis. The detection of analytes was achieved by laser-induced fluorescence in capillary electrophoresis. Procedures were validated according to the European Union regulation 2002/657/EC determining specificity, selectivity and detection capability for screening method and decision limit, detection capability, specificity, selectivity, trueness and precision for confirmation method. The results of validation process demonstrate that the method is suitable for application in Brazilian statutory veterinary drug residue surveillance programs. Capillary electrophoresis was proved to be a fast, robust method with low time and reagents consumption.     

A novel and rapid method for determination of natamycin in wines based on ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry: validation according to the 2002/657/EC European decision

A novel, simple, and rapid reversed-phase liquid chromatography–tandem mass spectrometric methodology was developed for the analysis of natamycin in wine samples. Natamycin was protonated to form singly charged ions in an electrospray positive ion mode. Data acquisition under MS/MS was achieved by applying multiple reaction monitoring (MRM) of three fragment ion transitions (666.3 → 648.2, 666.3 → 503.3, and 666.3 → 485.2) to provide a high degree of sensitivity and specificity. Chromatographic separation was performed on a rapid resolution column using a mobile phase consisting of an acetonitrile/water mixture with a total run time of 5.0 min. After only filtration as pretreatment, the sample was injected into the chromatographic system. The proposed method was validated in terms of selectivity, trueness, precision, decision limit (CCα), and detection capability (CCβ) according to 2002/657/EC Commission decision. The values for trueness, reported as bias (%), agreed with those established by the aforementioned document. Repeatability (intraday variability) values were 12.37% at a concentration of 1.0 μg L⁻¹ and 8.99–4.19% at concentrations between 2.5 and 10 μg L⁻¹. The overall within-laboratory (interday variability) reproducibility was 15.47% at a concentration of 1.0 μg L⁻¹, which was significantly lower than the indicative value reported in the EU decision. The results indicated that the proposed approach is a sensitive, fast, reproducible, and robust methodology suitable for the analysis of natamycin levels in wine samples.     

Validation of a procedure to quantify oxolinic acid,danofloxacin, ciprofloxacin and enrofloxacin in selected meats by micellar liquid chromatography according to EU Commission Decision 2002/657/EC

The suitability of an analytical method to determine oxolinic acid, danofloxacin, ciprofloxacin and enrofloxacin in edible tissues, based on micellar liquid chromatography coupled with fluorescence detection, to be applied in chicken, turkey, duck, lamb, goat, rabbit and horse muscle, is described. The method was fully matrix‐matched in‐lab revalidated, for each antimicrobial drug and meat, following the guidelines of the EU Commission Decision 2002/657/EC. The permitted limits were the maximum residue limits stated by the EU Commission Regulation 37/2010. The results obtained for the studied validation parameters were in agreement with the guidelines: selectivity (the antibiotics were resolved), linearity (r²  > 0.995), limit of detection (0.004–0.02 mg/kg), limits of quantification (0.01–0.05 mg/kg), calibration range (up to 0.5 mg/kg), recovery (89.5–105.0%), precision (<8.3%), decision limit, detection capability, ruggedness, stability and application to incurred samples. The method was found to be able to provide reliable concentrations with low uncertainty within a large interval, including the maximum residue limits, and then was useful to find out prohibited contaminated samples. The method did not require to be adapted for these matrices, and then it maintained its interesting advantages: short‐time, eco‐friendly, safe, inexpensive, easy‐to‐conduct, minimal manipulation and useful for routine analysis.     

Development of an analytical methodology to quantify melamine in milk using micellar liquid chromatography and validation according to EU Regulation 2002/654/EC

Melamine is a toxic triazine, illegally used as an additive in milk to apparently increase the amount of protein. A chromatographic procedure using a C18 column and a micellar mobile phase of sodium dodecyl sulphate (0.05 M) and propanol (7.5%), buffered at pH 3, and a detection set by absorbance at 210 nm, was reported for the resolution and quantification of melamine in liquid and powdered milk samples. In this work, samples were diluted with a SDS solution and were directly injected, thus avoiding long extraction and experimental procedures. Melamine was eluted in nearly 9.3 min without overlapping the protein band or other endogeneous compounds. The optimal mobile phase composition was taken using a chemometrical approach that considers the retention factor, efficiency and peak shape. Validation was performed following the European Commission's indications (European Decision 2002/657/EC), and the main analytical parameters studied were: linearity (0.02-100 ppm; r² = 0.999), limit of detection (5 ppb), intra- and inter-day precision (R.S.D. <7.6% and <9.7%, respectively) and robustness (R.S.D. <7.4% for retention time and <5.0% for area). Sensitivity was adequate to detect melamine under the safety limits proposed by the US FDA. Finally, recoveries for several milk samples were found in the 85-109% range.     

An ion-pairing liquid chromatography/tandem mass spectrometric method for the determination of ethephon residues in vegetables

A rapid, sensitive and selective method has been developed for the direct determination of ethephon residues in vegetables (apple, cherry and tomato). Given the anionic character of ethephon, the use of ion-pairing liquid chromatography (LC) in combination with tandem mass spectrometry (MS/MS, triple quadrupole) allowed its direct determination in these matrices avoiding a derivatisation step and favouring the automation of the method. Samples were extracted with a mixture of dichloromethane/aqueous formic acid (pH 3) (1:1). Then, tetrabutylammonium acetate (TBA) was added as an ion-pairing reagent, and an aliquot of the aqueous extract was directly injected into the LC/MS/MS system. Quantification was performed with matrix-matched standards prepared from blank sample extracts. MS/MS measurements were made in the selected reaction monitoring (SRM) mode, using the most sensitive transition (m/z 107 > 79) for quantification, and up to four additional transitions for confirmation. Quantitative recoveries were obtained for all matrices (between 83% and 96%) at two concentration levels tested (0.05 and 0.5 mg/kg), with relative standard deviations lower than 9% in all cases. The addition of TBA directly into the sample extract contained in the injection vial was found sufficient to obtain satisfactory LC retention for the analyte. Under these conditions, the absence of ion-pairing reagent in the mobile phase minimised the ionisation suppression for ethephon in the MS source, leading to an increase in the sensitivity of the method and reaching limits of detection of 0.02 mg/kg for all matrices investigated. The acquisition of five specific MS/MS transitions for ethephon allowed the simultaneous and reliable quantification and confirmation of the analyte in the samples.     

Multi-class, multi-residue liquid chromatography/tandem mass spectrometry screening and confirmation methods for drug residues in milk

This paper describes the development and optimization of a multi-residue veterinary drug screening method for whole milk. The drug residues of regulatory interest in milk include beta-lactams, sulfonamides, tetracyclines, fluoroquinolones, and macrolides. Milk samples were extracted with acetonitrile and the samples were then subjected to a clean-up procedure using a bonded solid-phase extraction cartridge and a molecular weight cut-off filter. Liquid chromatography/tandem mass spectrometry (LC/MS/MS) triple quadrupole electrospray methods were developed to monitor for the drugs in milk. Since established tolerance levels are set for most of these drugs in milk, the initial screening procedure was semi-quantitative, where samples were compared to the response of a positive control. The positive control, consisting of an extract from a portion of milk fortified with the drugs at half their allowed levels, was used to set the laboratory's minimum response criteria for unknown samples. Confirmatory analyses, with additional ion transitions for each residue, were performed on the same extracts.     

Bioanalytical applications of 'fast chromatography' to high-throughput liquid chromatography/tandem mass spectrometric quantitation

A fast chromatographic separation approach that enables rapid method development for high-throughput sample quantification is discussed. This approach has been used to analyze samples from various biological matrices. Data are presented from in vivo pharmacokinetic studies (plasma) and in vitro drug metabolism and transport studies (hepatic microsomes, hepatocytes, and Caco-2 cells).     

Multi-residue liquid chromatography/tandem mass spectrometric analysis of beta-agonists in urine using molecular imprinted polymers

Ion suppression, a matrix effect that affects quantitative mass spectrometry, is one of the main problems encountered in liquid chromatography/tandem mass spectrometry. Two different clean-up steps for the multi-residue analysis of beta-agonists in urine were evaluated with respect to minimisation of ion suppression, namely, a mixed-phase solid phase extraction (SPE) column, i.e., clean screen Dau (CSD), and a molecular imprinted polymer (MIP) SPE column. Ion suppression experiments revealed that CSD sample clean-up can lead to false negative results for some beta-agonists, and that clean-up using MIP columns is more selective for beta-agonists than the use of CSD columns.     

Development and validation of an HPLC method for the determination of ten sulfonamide residues in milk according to 2002/657/EC

A HPLC method with diode-array detection, at 265 nm, was developed and validated for the determination of ten sulfonamides (SAs): sulfadiazine (SDZ), sulfathiazine (STZ), sulfamethoxine (SMTH), sulfamethizole (SMZ), sulfamethoxypyridazine (SMPZ), sulfamonomethoxine (SMMX), sulfamethoxazole (SMXZ), sulfisoxazole (SIX), sulfadimethoxine (SDMX), and sulfaquinoxaline (SQX) in milk. A mixture of ethyl acetate, n-hexane, and isopropanol was used for the extraction of target analytes from milk. The mobile phase, a mixture of 0.1% v/v formic acid, CH(3) CN, and CH(3) OH was delivered to the analytical column under a gradient program. The procedure was validated according to the European Union regulation 2002/657/EC in terms of selectivity, stability, decision limit, detection capability, accuracy, and precision. Mean recoveries of sulfonamides from milk samples spiked at three concentration levels (0.5×MRL, 1×MRL, and 1.5×MRL) (MRL, maximum residue level) were 93.9-115.9% for SDZ, 97.8-102.9% for STZ, 94.6-107.0% for SMTH, 98.3-111.5% for SMZ, 95.3-108.4% for SMPZ, 97.9-106.0% for SMMX, 97.6-111.3% for SMXZ, 94.3-104.6% for SIX, 96.4-109.1% for SDMX, and 98.2-111.2% for SQX. All RSD values were lower than 8.8%. The decision limits CCa calculated by spiking 20 blank milk samples at MRL (100 μg/kg) ranged from 101.61 to 106.84 μg/kg, whereas the detection capability CCb ranged from 105.64 to 119.01 μg/kg.     

Optimization and validation of a multiclass screening and confirmation method for drug residues in milk using high-performance liquid chromatography/tandem mass spectrometry

The further optimization and validation of a multiresidue veterinary drug screening method for milk is described. The drug residues of regulatory interest in milk include beta-lactams, sulfonamides, tetracyclines, fluoroquinolones, and macrolides. A previously published procedure has been modified to incorporate new compounds and to collect both screening and confirmatory ion transitions in one acquisition method. Milk samples were extracted with an equal volume of acetonitrile. The samples were then subjected to cleanup with a bonded SPE cartridge and a MW cutoff filter. The SPE protocol was modified to effectively recover a metabolite of flunixin. Established tolerance levels are set for most of these drugs in milk; thus, the screening procedure was semiquantitative, using positive controls for comparison. The positive controls, consisting of extracts from milk fortified with the drugs at their tolerance or safe level, were used to set statistically valid minimum response criteria for unknown samples. This updated method was validated with fortified milk, as well as with milk samples from animals administered veterinary drugs.     

Use of liquid chromatography/tandem mass spectrometric molecular fingerprinting for the rapid structural identification of pharmaceutical impurities

Use of liquid chromatography/tandem mass spectrometric (LC/MSⁿ) molecular fingerprinting is systematically demonstrated as a very effective tool for rapid structural elucidation of pharmaceutical impurities through a case study in which three isomers of betamethasone sodium phosphate (BSP) were rapidly identified as degradants formed due to the D‐homoannular ring expansion of the steroid core structure of BSP in the solid state. The rapid structural elucidation of these degradants was achieved by matching or closely matching the UV profiles, molecular weights, and more importantly the fragmentation patterns obtained from the LC/MSⁿ (n = 1 to 3) analysis of their enzyme‐catalyzed hydrolytic products, respectively, with those of a D‐homoannular isomer of betamethasone available in our LC/MSⁿ molecular fingerprint database. This strategy of using LC/MSⁿ molecular fingerprinting to obtain high‐confidence structures of unknown species is then validated by structure verification through one‐ (1D) and two‐dimensional (2D) nuclear magnetic resonance (NMR) experiments. Copyright © 2009 John Wiley & Sons, Ltd.     

Multiresidue analysis of sulfonamides in meat by supramolecular solvent microextraction,liquid chromatography and fluorescence detection and method validation according to the 2002/657/EC decision

A multiresidue method was described for determining eight sulfonamides, SAs (sulfadiazine, sulfamerazine, sulfamethoxypyridazine, sulfachloropyridazine, sulfadoxine, sulfamethoxazole, sulfadimethoxine and sulfaquinoxaline) in animal muscle tissues (pork, chicken, turkey, lamb and beef) at concentrations below the maximum residue limit (100 μg kg⁻¹) set by the European Commission. The method was based on the microextraction of SAs in 300-mg muscle samples with 1 mL of a supramolecular solvent made up of reverse micelles of decanoic acid (DeA) and posterior determination of SAs in the extract by LC/fluorescence detection, after in situ derivatization with fluorescamine. Recoveries were quantitative (98–109%) and matrix-independent, no concentration of the extracts was required, the microextraction took about 30 min and several samples could be simultaneously treated. Formation of multiple hydrogen bonds between the carboxylic groups of the solvent and the target SAs (hydrogen donor and acceptor sum between 9 and 11) were considered as the major forces driving microextraction. The method was validated according to the European Union regulation 2002/657/EC. Analytical performance in terms of linearity, selectivity, trueness, precision, stability of SAs, decision limit and detection capability were determined. Quantitation limits for the different SAs ranged between 12 μg kg⁻¹ and 44 μg kg⁻¹, they being nearly independent of matrix composition. Repeatability and reproducibility, expressed as relative standard deviation, were in the ranges 1.8–3.6% and 3.3–6.1%. The results of the validation process proved that the method is suitable for determining sulfonamide residues in surveillance programs.     

Importance of using highly pure internal standards for successful liquid chromatography/tandem mass spectrometric bioanalytical assays

Internal standards (IS) with similar physicochemical properties to the analyte provide multiple advantages in liquid chromatography/tandem mass spectrometric (LC/MS/MS) bioanalytical methods such as: reduction of the analysis run time, improvement in the intra‐injection reproducibility, impact reduction of matrix and ionization effects. However, it is important to evaluate the purity of the IS prior to their use. Indeed, a minor impurity in the IS may lead to an important issue during bioanalytical method development. Stable labelled internal standards are usually appropriate IS for bioanalysis. The use of oxycodone‐D3, ursodiol‐D5 and atovaquone‐D4 as internal standards in three different bioanalytical methods was evaluated. During oxycodone, oxymorphone and noroxycodone simultaneous quantification method development, oxymorphone was identified as a contaminant in oxycodone‐D3. Since the limit of quantification for oxymorphone was very low (10 pg/mL), the presence of an even low percentage of oxymorphone in oxycodone‐D3 leads to the change of the stable labelled IS for an analogue, ethylmorphine. 23‐Nordeoxycholic acid was preferred to ursodiol‐D5 as internal standard for the ursodiol, tauroursodiol and glycoursodiol simultaneous quantification method. Indeed, more than 7% of ursodiol was identified in the ursodiol‐D5 which could not be bypassed by decreasing the IS concentration without compromising the linearity. An atovaquone‐D4 reference standard revealed the non‐negligible presence of atovaquone‐D5 to atovaquone‐D8 that has a large impact on the method validation. Therefore, atovaquone‐D4 was sent for recertification since its isotopic purity was found to be much less than the isotopic purity mentioned on its certificate of analysis. Consequently, during bioanalytical method development, the purity of the IS should be scrutinized. Copyright © 2009 John Wiley & Sons, Ltd.     

Structural identification of mouse urinary metabolites of pterostilbene using liquid chromatography/tandem mass spectrometry

Pterostilbene, the dimethoxy derivative of resveratrol, has drawn much attention recently due to its potential beneficial health effects. The metabolic fate of pterostilbene, however, is not well understood. In the present study, we identified nine novel mouse urinary pterostilbene metabolites, pterostilbene glucuronide, pterostilbene sulfate, mono‐demethylated pterostilbene glucuronide, mono‐demethylated pterostilbene sulfate, mono‐hydroxylated pterostilbene, mono‐hydroxylated pterostilbene glucuronide, mono‐hydroxylated pterostilbene sulfate, and mono‐hydroxylated pterostilbene glucuronide sulfate, using liquid chromatography/atmospheric pressure chemical ionization and electrospray ionization tandem mass spectrometry. The structures of these metabolites were confirmed by analyzing the MSⁿ (n = 1–3) spectra. To our knowledge, this is the first report of the identification of urinary metabolites of pterostilbene in mice. Copyright © 2010 John Wiley & Sons, Ltd.     

Microcapillary liquid chromatography/tandem mass spectrometry using alkaline pH mobile phases and positive ion detection

Extending the dynamic range of microcapillary liquid chromatography/tandem mass spectrometry (LC/MS/MS) peptide sequencing methods is essential for extracting new discoveries from proteomic studies. The complexity of global protein digests and in vivo processed peptide repertoires (as isolated from immunologically important HLA complexes) have led to the development of novel separation methods to increase the number of peptides identified by a single analysis. Separation of complex mixtures by multidimensional high-performance liquid chromatography (HPLC) decreases the number of isolated peptides contained in each fraction and increases the likelihood of detecting low abundant peptides in a background of dominant signals. In this study, we have evaluated the use of two dimensions of reversed-phase chromatography for resolving and sequencing naturally processed HLA-A2 presented peptide repertoires. The first dimension of separation was reversed-phase chromatography using the strong ion pairing reagent trifluoroacetic acid (TFA) to ensure the highest efficiency of peptide fractionation. The second dimension of reversed-phase chromatography was online with an electrospray ionization (ESI) ion trap mass spectrometer. Mobile phases used for the second dimension of chromatography were modified with volatile reagents including a contemporary acetate-modified acidic solvent, which was compared with mobile phases prepared with ammonium hydroxide at an alkaline pH. As expected, we demonstrate improved separation of the HLA-A2 presented fractions using the alkaline pH conditions. However, less obvious was the improved peptide signal-to-noise detected for peptide signals by positive ion ESI ion trap mass spectrometric detection, which was attributed to a reduced chemical background when using the alkaline pH mobile phases that allowed the ion trap to fill with the peptide ions until the automatic gain control detected a full trap. The term 'wrong-way-round ionization' has been used to describe intense [M+H](+) ions generated during ESI under strongly basic solutions. Ultimately, a larger number of the HLA-A2 peptide repertoire was sequenced by coupling TFA-modified reversed-phase fractionation with alkaline-modified microcapillary LC/MS/MS analysis of each fraction. In the present report, we compare the two second-dimension approaches and demonstrate the quality of data that was acquired using alkaline pH reversed-phase conditions.     

A new multi-residue method for analysis of pesticide residues in fruit and vegetables using liquid chromatography with tandem mass spectrometric detection

A new multi-residue method for determination of pesticide residues in a wide variety of fruit and vegetables, using the National Food Administration (NFA) ethyl acetate extraction and determination by means of LC-MS/MS, is presented. The method includes pesticides normally detected by LC-UV or LC-fluorescence such as benzimidazoles, carbamates, N-methylcarbamates and organophosphorus compounds with an oxidisable sulphide group as well. After extraction with ethyl acetate, the extract is concentrated and an aliquot of the extract is evaporated to dryness and redissolved in methanol before injection on LC-MS/MS. The method has been validated for 57 different pesticides and metabolites. Representative species from different commodity groups were chosen as matrices in order to study the influence from different matrices on recoveries. The fortification levels studied were 0.01-0.5 mg kg(-1). Matrix effects were tested for all matrices by means of standard addition to blank extracts. The matrix effect, expressed as signal in solvent compared to signal in matrix, was in general found to be small. The obtained recoveries are, with a few exceptions, in the range 70-100%. The proposed method is quick and straightforward and no additional clean-up steps are needed. The method can be used for the analysis of all 57 pesticides in one single determination step at 0.01 mg kg(-1).     

Determination and confirmation of tulathromycin residues in bovine liver and porcine kidney via their common hydrolytic fragment using high-performance liquid chromatography/tandem mass spectrometry

An accurate, reliable, and reproducible analytical method using HPLC/MS/MS for the determination of tulathromycin residues in bovine liver and porcine kidney via their common hydrolytic fragment (CP-60,300) was developed and validated. Briefly, the method involved an initial acid treatment of intact tissues, which yielded the common fragment (CP-60,300). A portion of the acid hydrolyzate was purified by SPE using a strong cation exchange cartridge. Evaporation of the purified extract was followed by reconstitution in aqueous buffer and analysis by HPLC/MS/MS under isocratic conditions. The developed method provided acceptable sensitivity for determinative surveillance of tulathromycin in porcine kidney and bovine liver with an LOQ of 7.50 and 2.75 microg/g, respectively. The overall recovery and precision of 45 determinations of each tissue were 97.8% (5.3%) for porcine kidney and 96.9% (7.9%) for bovine liver. Accuracy, precision, linearity, specificity, and ruggedness were demonstrated. An HPLC/MS/MS method was also developed for use in these tissues as a confirmatory assay following modifications to the MS detection parameters. The confirmatory method demonstrated acceptable sensitivity for confirmatory evaluation of tulathromycin in porcine kidney and bovine liver at tolerances of 15 and 5.5 microg/g, respectively.     

Automated nanoflow liquid chromatography/tandem mass spectrometric identification of proteins from Shewanella putrefaciens separated by two-dimensional polyacrylamide gel electrophoresis

The implementation of nanoflow liquid chromatography offers unique opportunities for automation of proteomics research. We demonstrate that automated nanoflow LC/MS/MS allowed the unambiguous identification of proteins from the omnipotent bacterium Shewanella putrefaciens, based on similarity searches against the completely determined genome of related microorganisms and against non-redundant databases. Total protein extracts were separated by 2-dimensional polyacrylamide electrophoresis. Only 1/20th of a tryptic digest mixture obtained from a single Coomassie Blue stained spot was loaded on the nanoflow LC column using a preconcentration/desalting step, and analyzed on-line on a hybrid quadrupole time-of-flight mass spectrometer with an automated MS-to-MS/MS switching protocol. This method allowed the de novo peptide sequence determination of several tryptic fragments and the identification of different proteins. CopyrightCopyright 2000 John Wiley & Sons, Ltd.