Electrospray Interface Research Articles

Capillary zone electrophoresis-multiple reaction monitoring from 100 pg of RAW 264.7 cell lysate digest

Capillary zone electrophoresis-multiple/single reaction monitoring (CZE-MRM/SRM), which employed an electrokinetically driven sheath-flow electrospray interface, was used for the rapid and highly sensitive detection of protein analytes in complex tryptic digests. MRM channels were developed against a commercial exponential mixture of bovine proteins. Five proteins spanning four orders of magnitude concentration range were confidently detected from only 2.5 ng of the digest mixture; the mass detection limits (S/N = 3) of two detected proteins, alpha-casein and glutamate dehydrogenase were about 600 zmol and 30 amol, respectively. This technique was then applied to a RAW 264.7 cell lysate digest. Three proteins were confidently and reproducibly detected from 100 pg of this digest. The sample amount corresponds to the approximate protein content from a single cell, which suggests that CZE-MRM may be a useful analytical tool in chemical cytometry. In addition to providing highly sensitive detection of proteins in complex mixtures, this system is highly rapid; migration time of the protein digests was less than 10 min.

Highly sensitive ultra-performance liquid chromatography–tandem mass spectrometry method for the determination of abiraterone in human plasma

Abiraterone (ABR) is a rationally designed CYP17 inhibitor that blocks the conversion of androgens from non-gonadal precursors effectively, thus reducing testosterone to undetectable levels. With the objective of reducing analysis time and maintaining good efficiency, there has been substantial focus on high-speed chromatographic separations, and ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) is a pre-eminent analytical tool for rapid biomedical analysis. In this study a rapid and precise UPLC–MS/MS method has been developed and validated for the determination of ABR in plasma. After a simple protein precipitation using methanol, ABR and carbamazepine (internal standard; IS) were separated on an Acquity UPLC BEH™ C18 column (50 × 2.1 mm, i.d. 1.7 μm, Waters, USA) using a mobile phase of acetonitrile : water : formic acid (90 : 10 : 0.1%, v/v/v) pumped at a flow rate of 0.3 mL min−1. ABR and IS were eluted at 0.61 and 0.48 min, respectively. The mass spectrometric determination was carried out using an electrospray interface operated in the positive mode with multiple reaction monitoring (MRM) mode. The precursors to product ion transitions of m/z 350.1 > 156.0, and m/z 237.0 > 179.0 were used to quantify ABR and IS, respectively and m/z 350.1 > 170.0 transition was used as the qualifying ion for ABR. The method was linear in the concentration range of 0.1–50 ng mL−1 with good correlation coefficient of (0.995) and with a limit of quantitation of 0.1 ng mL−1. The intra- and inter-assay precisions were satisfactory; the relative standard deviations did not exceed 13.29%. The accuracy of the method was proved; recoveries of ABR from spiked human plasma were 75.83–78.33%. The proposed UPLC–MS/MS method is simple, rapid and highly sensitive, and hence it could be reliable for pharmacokinetic and toxicokinetic studies in both animals and humans.

Determination of malachite green, crystal violet and their leuco-metabolites in fish by HPLC–VIS detection after immunoaffinity column clean-up

A high performance liquid chromatography method with visible detection (HPLC–VIS) for the determination of malachite green (MG), crystal violet (CV), leucomalachite green (LMG), and leucocrystal violet (LCV) in fish has been developed after clean-up through an immunoaffinity column (IAC). Residues were simultaneously extracted from fish muscle with acetonitrile and ammonium acetate buffer. The leuco-forms, LMG and LCV, were oxidized quantitatively to the chromic CV and MG by reaction with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone. Extracts were then purified on an IAC which prepared by immobilizing the anti-MG–CV antibodies by the sol–gel method. Finally, the eluents were analyzed by HPLC–VIS. The limits of detection were 0.15, 0.1, 0.18 and 0.14ng/g for MG, CV, LMG and LCV, respectively. The average recoveries in samples fortified with MG, CV, LMG and LCV over the range 0.5–10ng/g were from 71.6% to 96.8% with RSDs of 5.1–12.3% (n=6). This novel method was confirmed by liquid chromatography–tandem mass spectrometry with electrospray interface in positive mode using multiple reaction monitoring.

Evaluation of a direct high-capacity target screening approach for urine drug testing using liquid chromatography–time-of-flight mass spectrometry

In this study a rapid liquid chromatography–time-of-flight mass spectrometry method was developed, validated and applied in order to evaluate the potential of this technique for routine urine drug testing. Approximately 800 authentic patient samples were analyzed for amphetamines (amphetamine and methamphetamine), opiates (morphine, morphine-3-glucuronide, morphine-6-glucuronide, codeine and codeine-6-glucuronide) and buprenorphines (buprenorphine and buprenorphine-glucuronide) using immunochemical screening assays and mass spectrometry confirmation methods for comparison. The chromatographic application utilized a rapid gradient with high flow and a reversed phase column with 1.8μm particles. Total analysis time was 4min. The mass spectrometer operated with an electrospray interface in positive mode with a resolution power of >10,000 at m/z 956. The applied reporting limits were 100ng/mL for amphetamines and opiates, and 5ng/mL for buprenorphines, with lower limits of quantification were 2.8–41ng/mL. Calibration curves showed a linear response with coefficients of correlation of 0.97–0.99. The intra- and interday imprecision in quantification at the reporting limits were <10% for all analytes but for buprenorphines <20%. Method validation data met performance criteria for a qualitative and quantitative method. The liquid chromatography–time-of-flight mass spectrometry method was found to be more selective than the immunochemical method by producing lower rates of false positives (0% for amphetamines and opiates; 3.2% for buprenorphines) and negatives (1.8% for amphetamines; 0.6% for opiates; 0% for buprenorphines). The overall agreement between the two screening methods was between 94.2 and 97.4%. Comparison of data with the confirmation (LC–MS) results for all individual 9 analytes showed that most deviating results were produced in samples with low levels of analytes. False negatives were mainly related to failure of detected peak to meet mass accuracy criteria (±20mDa). False positives was related to presence of interfering peaks meeting mass accuracy and retention time criteria and occurred mainly at low levels. It is concluded that liquid chromatography–time-of-flight mass spectrometry has potential both as a complement and as replacement of immunochemical screening assays.

A surrogate analyte method to determine d-serine in mouse brain using liquid chromatography–tandem mass spectrometry

A bioanalytical method for determining endogenous d-serine levels in the mouse brain using a surrogate analyte and liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed. [2,3,3-2H]d-serine and [15N]d-serine were used as a surrogate analyte and an internal standard, respectively. The surrogate analyte was spiked into brain homogenate to yield calibration standards and quality control (QC) samples. Both endogenous and surrogate analytes were extracted using protein precipitation followed by solid phase extraction. Enantiomeric separation was achieved on a chiral crown ether column with an analysis time of only 6min without any derivatization. The column eluent was introduced into an electrospray interface of a triple–quadrupole mass spectrometer. The calibration range was 1.00 to 300nmol/g, and the method showed acceptable accuracy and precision at all QC concentration levels from a validation point of view. In addition, the brain d-serine levels of normal mice determined using this method were the same as those obtained by a standard addition method, which is time-consuming but is often used for the accurate measurement of endogenous substances. Thus, this surrogate analyte method should be applicable to the measurement of d-serine levels as a potential biomarker for monitoring certain effects of drug candidates on the central nervous system.

Comparing Procyanidins in Selected Vaccinium Species by UHPLC-MS2 with Regard to Authenticity and Health Effects

Cranberry procyanidins have been associated with an effect against urinary tract infections (UTI) for decades, and European health claims are requested. This study compares the procyanidin profiles and concentrations of American cranberry ( Vaccinium macrocarpon Ait.), European cranberry ( Vaccinium oxycoccus L.), and lingonberry ( Vaccinium vitis-idaea L.) analyzed using ultrahigh-performance liquid chromatoraphy coupled to a triple-quadrupole mass spectrometer with electrospray interface (UHPLC-MS(2)). Concentrations of A-type trimers, procyanidin A2, catechin, epicatechin, and B-type dimers and trimers have been evaluated and compared for the first time in the three berries. The data clearly show remarkable differences in the procyanidin profiles and concentrations, especially the lack of A-type trimers in V. oxycoccus; thus, the effectiveness against UTI may vary among the Vaccinium species. These differences can be used to prove authenticity.

Method development and validation for the quantification of dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, sorafenib and sunitinib in human plasma by liquid chromatography coupled with tandem mass spectrometry

To support pharmacokinetic-guided dosing in individual patients, a fast and accurate method for simultaneous determination of anticancer tyrosine kinase inhibitors (TKIs) dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, sorafenib and sunitinib in human plasma was developed using high-performance liquid chromatography and detection with tandem mass spectrometry (HPLC-MS/MS). Stable isotopically labeled compounds of the eight different TKIs were used as internal standards. Plasma proteins were precipitated and an aliquot of supernatant was directly injected onto a reversed phase chromatography system consisting of a Gemini C18 column (50 × 2.0 mm i.d., 5.0 µm particle size) and then compounds were eluted with a gradient. The outlet of the column was connected to a triple quadrupole mass spectrometer with electrospray interface. Ions were detected in the positive multiple reaction monitoring mode. This method was validated over a linear range from 20.0 to 10,000 ng/mL for erlotinib, gefitinib, imatinib, lapatinib, nilotinib and sorafenib, and from 5.00 to 2500 ng/mL for dasatinib and sunitinib. Results from the validation study demonstrated good intra- and inter-assay accuracy (<13.1%) and precision (10.0%) for all analytes. This method was successfully applied for routine therapeutic drug monitoring purposes in patients treated with the investigated TKIs.

CZE‐ESI‐MS/MS system for analysis of subnanogram amounts of tryptic digests of a cellular homogenate

We report the performance of capillary zone electrophoresis coupled with an electrokinetically pumped electrospray interface and an Orbitrap-Velos mass spectrometer for high sensitivity protein analysis. We first investigated the system for quantitation of the tryptic digest of BSA. The system produced outstanding linearity with respect to peak height, number of peptide IDs, and spectral counts across the range of 12 nM to 750 nM (60 amol to 3.5 fmol) of BSA injected. One peptide produced a detection limit of 0.3 nM (1.5 amol) injected. We also analyzed 700 pg of a tryptic digest prepared from a RAW264.7 cell lysate; ten proteins were identified in triplicate analyses after filtering the data with peptide confidence value as high. This sample size corresponds to the protein content of approximately ten eukaryotic cells.

Liquid chromatography–tandem mass spectrometric assay for the ALK inhibitor crizotinib in mouse plasma

A quantitative bioanalytical liquid chromatography–tandem mass spectrometric (LC–MS/MS) assay for the ALK inhibitor crizotinib was developed and validated. Plasma samples were pre-treated using protein precipitation with acetonitrile containing crizotinib-13C2-2H5 as internal standard. The extract was directly injected into the chromatographic system after dilution with water. This system consisted of a sub-2μm particle, trifunctional bonded octadecyl silica column with a gradient using 0.1% (v/v) of ammonium hydroxide in water and methanol. The eluate was transferred into the electrospray interface with positive ionization and the analyte was detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was validated in a 10–10,000ng/ml calibration range with r2=0.99980±0.00014 for double logarithmic linear regression (n=5). Within day precisions (n=6) were 3.4–4.8%, between day (3 days; n=18) precisions 3.6–4.9%. Accuracies were between 107% and 112% for the whole calibration range. The drug was sufficiently stable under all relevant analytical conditions. Oxidative metabolites of crizotinib were monitored semi-quantitatively. Finally, the assay was successfully used to assess drug pharmacokinetics in mice.

Liquid chromatography–tandem mass spectrometric assay for therapeutic drug monitoring of the tyrosine kinase inhibitor pazopanib in human plasma

A quantitative bioanalytical liquid chromatography–tandem mass spectrometric assay for the tyrosine kinase inhibitor pazopanib was developed and validated. Plasma samples were pre-treated using protein precipitation with acetonitrile containing pazopanib-d4 as internal standard. The extract was injected into the chromatographic system after dilution with water (1:9, v/v). The system consisted of a sub-2μm particle, trifunctional bonded octadecyl silica column with isocratic elution using 0.005% (v/v) of formic acid in a mixture of water (76%, v/v) and acetonitrile (24%, v/v). The analyte was quantified using the selected reaction monitoring mode of a triple quadrupole mass spectrometer with a heated electrospray interface. The assay was validated in a 0.1–100μg/ml calibration range. Within day precisions were 3.6–5.2%, between day precisions 4.0–8.3% and accuracies between 106% and 113% for the whole calibration range. The drug was sufficiently stable under all relevant analytical conditions. The assay has successfully been used to assess drug levels for therapeutic drug monitoring in patients treated with pazopanib.

Quantitative multiple reaction monitoring of peptide abundance introduced via a capillary zone electrophoresis-electrospray interface.

We demonstrate the use of capillary zone electrophoresis with an electrokinetic sheath-flow electrospray interface coupled to a triple-quadrupole mass spectrometer for the accurate and precise quantification of Leu-enkephalin in a complex mixture using multiple-reaction monitoring (MRM). Assay time is <6 min, with no re-equilibration required between runs. A standard curve of Leu-enkephalin was performed in the presence of a background tryptic digest of bovine albumin. We demonstrate reasonably reproducible peak heights (21% relative standard deviation), retention times (better than 1% relative standard deviation), and robust electrospray quality. Our limit of detection (3σ) was 60 pM, which corresponds to the injection of 335 zmol of peptide. This is a 10-20-fold improvement in mass sensitivity than we have obtained by nano HPLC/MRM and substantially better than reported for LC/MS/MS. Further quantification was performed in the presence of stable-isotope-labeled versions of the peptides; under these conditions, linearity was observed across nearly 4 orders of magnitude. The concentration detection limit was 240 pM for the stable-isotope-labeled quantification.

Determination of alcohol sulfates and alcohol ethoxysulfates in wastewater samples by liquid chromatography tandem mass spectrometry

The work develops a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the identification and quantification of alkyl homologues of alcohol sulfates (AS) and ethoxymers of alcohol ethoxysulfates (AES) in wastewaters. The method involves isolation and preconcentration of the analytes from the samples using solid-phase extraction (SPE) with SAX cartridges prior to their quantification by LC–MS/MS with electrospray (ESI) interface in negative mode. The limits of detection (LOD) found were 0.1μgL−1 for AS, and ranged from 0.2 to 0.5μgL−1 for AES. The limits of quantification (LOQ) ranged from 0.3 to 0.4μgL−1 for AS, and from 0.5 to 1.5μgL−1 for AES. The accuracy of the method was studied in terms of trueness and precision. Trueness was evaluated by using a spike recovery assay with spiked blank samples. Recoveries ranged from 96.0 to 102.8% for AS and from 93.4 to 105.1% for AES. Precision (in terms of inter- and intra-day RSD) was lower than 5% for AS and AES. Finally, the method was satisfactorily applied to the quantification of compounds in wastewater samples collected from a wastewater treatment plant (WWTP) located in the city of Granada (Spain). Data obtained showed that AS are present in concentration levels from 0.7 to 39.4μgL−1 in influent waters and from 1.1 to 8.1μgL−1 in effluent waters. As for AES, the amounts (sum of ethoxymers) found ranged from 0.7 to 24.0μgL−1 in influents and from 1.9 to 2.5μgL−1 in effluents.

Liquid chromatography–tandem mass spectrometric assay for the VEGFR inhibitor cediranib and its primary human metabolite cediranib-N+-glucuronide in plasma

A quantitative bioanalytical assay for cediranib and its N+-glucuronide metabolite was developed and validated. Human plasma samples were pre-treated using protein precipitation with acetonitrile containing erlotinib and CYT-387 as internal standards for the glucuronide metabolite and parent compound, respectively. The extract was diluted with water and injected into the chromatographic system. This system consisted of sub-2μm particles, a trifunctional bonded octadecyl silica column with gradient elution using 0.005% (v/v) of formic acid in a mixture of water and methanol. The eluate was transferred into the electrospray interface with positive ionization and the analytes were detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was validated in a 1–290ng/ml calibration range for cediranib and 0.2–52ng/ml for its glucuronide metabolite. The lowest levels of these ranges corresponded to the lower limits of quantification for both compounds. Within day precisions were 4.0–6.7% for cediranib and 4.1–11.9% for its glucuronide, between day precisions were 4.2–10.2 and 4.8–14.4% and accuracies were between 99 and 106 and 84 and 94% for cediranib and its metabolite, respectively. Stabilities of both compounds were sufficient under all relevant conditions. Finally, the assay was successfully used to assess drug levels in a pharmacokinetic mouse study.

Liquid chromatography–tandem mass spectrometric assay for the JAK2 inhibitor CYT387 in plasma

A quantitative bioanalytical liquid chromatography–tandem mass spectrometric (LC–MS/MS) assay for the JAK2 inhibitor CYT387 was developed and validated. Plasma samples were pre-treated using protein precipitation with acetonitrile containing cediranib as internal standard. The extract was directly injected into the chromatographic system after dilution with water. This system consisted of a sub-2μm particle, trifunctional bonded octadecyl silica column with a gradient using 0.005% (v/v) of formic acid in a mixture of water and methanol. The eluate was transferred into the electrospray interface with positive ionization and the analyte was detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was validated in a 0.25–1000ng/ml calibration range. Within day precisions were 3.0–13.5%, between day precisions 5.7% and 14.5%. Accuracies were between 96% and 113% for the whole calibration range. The drug was stable under all relevant analytical conditions. Finally, the assay was successfully used to assess drug levels in mice.

Artificial Oxidation of Cysteine Residues in Peroxiredoxin 6 Detected by Twodimensional Gel Electrophoresis and Capillary Liquid Chromatography-Electrospray Mass Spectrometry

Artificially oxidized cysteine residues in peroxiredoxin 6 (Prx6) were detected by electrospray interface capillary liquidchromatography-linear ion trap mass spectrometry after the preparation of two-dimensional gel electrophoresis (2D-GE). Weused Prx6 as a model protein because it possesses only two cysteine residues at the 47th and 91st positions. The spot of Prx6 on2D-GE undergoes a basic (isoelectric point, pI 6.6) to acidic (pI 6.2) shift by exposure to peroxide due to selective overoxidationof the active-site cysteine Cys-47 but not of Cys-91. However, we detected a tryptic peptide containing cysteine sulfonic acid atthe 47th position from the basic spot and a peptide containing both oxidized Cys-47 and oxidized Cys-91 from the acidic spot ofPrx6 after the separation by 2D-GE. We prepared two types of oxidized Prx6s: carrying oxidized Cys-47 (single oxidized Prx6),and other carrying both oxidized Cys-47 and Cys-91 (double oxidized Prx6). Using these oxidized Prx6s, the single oxidizedPrx6 and double oxidized Prx6 migrated to pIs at 6.2 and 5.9, respectively. These results suggest that oxidized Cys-47 from thebasic spot and oxidized Cys-91 from the acidic spot are generated by artificial oxidation during sample handling processes afterisoelectric focusing of 2D-GE. Therefore, it is important to make sure of the origin of cysteine oxidation, if it is physiological orartificial, when an oxidized cysteine residue(s) is identified.

Liquid chromatography–tandem mass spectrometric assay for the mutated BRAF inhibitor vemurafenib in human and mouse plasma

A bioanalytical assay for the mutated BRAF inhibitor vemurafenib was developed and validated. For the quantitative assay, human plasma samples were pre-treated using protein precipitation with water-acetonitrile (1/3, v/v) containing sorafenib as internal standard. The extract was directly injected into the chromatographic system. This system consisted of a sub-2μm particle, trifunctional bonded octadecyl silica column with isocratic elution using 0.01% (v/v) of formic acid in a mixture of water and methanol. The eluate was transferred into the electrospray interface with positive ionization and the analyte was detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was validated in a 0.1–100μg/ml calibration range. Within day precisions were 1.6–3.2%, between day precisions 2.7% and 8.2% and accuracies were between 99% and 106% for the whole calibration range. The drug was stable under all relevant conditions. Finally, the assay was successfully used to assess drug levels in a pharmacokinetic mouse study.

Determination and confirmation of chloramphenicol in honey, fish and prawns by liquid chromatography–tandem mass spectrometry with minimum sample preparation: validation according to 2002/657/EC Directive

A reliable, simple and sensitive liquid chromatography–electrospray ionisation-tandem mass spectrometry (LC–ESI-MS/MS) confirmation method has been developed for chloramphenicol (CAP) determination in honey, fish and prawns. For honey, samples were extracted with ethyl acetate, an aliquot was evaporated to dryness and re-dissolved in mobile phase. For fish and prawns, tissues were extracted with acetonitrile and chloroform. The organic layer was evaporated to dryness and the residue was re-constituted with water: acetonitrile (90:10). LC separation was achieved on a C18 column with gradient elution using a mobile phase of acetonitrile and water. Analysis was carried out on a triple–quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode via electrospray interface operated in negative ionisation mode, with deuterated chloramphenicol-d5 (d5-CAP) as internal standard. Method validation was performed according to the criteria of Commission Decision 2002/657/EC. Four identification points were obtained for CAP with one precursor ion and two product ions. The limit of detection (LOD) was 0.02 µg kg−1. Linear calibration curves were obtained over concentration ranges of 0.1–1.0 µg kg−1 in tissues. Mean recoveries ranged from 85.5% to 115.6%, with the corresponding intra- and inter-day variation ranging from 1.0% to 22.5%, depending on matrix type and level of concentration. The decision limit (CCα) and detection capability (CCβ) of the method were obtained for all matrices: 0.04 and 0.06 µg kg−1, respectively, for prawns and fish and 0.05 and 0.09 µg kg−1 for honey.

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry as an alternative proteomics platform to ultraperformance liquid chromatography-electrospray ionization-tandem mass spectrometry for samples of intermediate complexity.

We demonstrate the use of capillary zone electrophoresis with an electrokinetically pumped sheath-flow electrospray interface for the analysis of a tryptic digest of a sample of intermediate protein complexity, the secreted protein fraction of Mycobacterium marinum. For electrophoretic analysis, 11 fractions were generated from the sample using reverse-phase liquid chromatography; each fraction was analyzed by CZE-ESI-MS/MS, and 334 peptides corresponding to 140 proteins were identified in 165 min of mass spectrometer time at 95% confidence (FDR < 0.15%). In comparison, 388 peptides corresponding to 134 proteins were identified in 180 min of mass spectrometer time by triplicate UPLC-ESI-MS/MS analyses, each using 250 ng of the unfractionated peptide mixture, at 95% confidence (FDR < 0.15%). Overall, 62% of peptides identified in CZE-ESI-MS/MS and 67% in UPLC-ESI-MS/MS were unique. CZE-ESI-MS/MS favored basic and hydrophilic peptides with low molecular masses. Combining the two data sets increased the number of unique peptides by 53%. Our approach identified more than twice as many proteins as the previous record for capillary electrophoresis proteome analysis. CE-ESI-MS/MS is a useful tool for the analysis of proteome samples of intermediate complexity.

Determination of amino acids and amines in mammalian decomposition fluid by direct injection liquid chromatography-electrospray ionisation-tandem mass spectrometry

A sensitive and selective analytical method utilising liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS) operated in multiple reaction monitoring mode was developed for the semi-quantitative determination of 19 biogenic amines and amino acids in mammalian (porcine) decomposition fluid. The effect of the matrix on analyte response was initially investigated by diluting crude samples and by injecting various volumes in the LC-MS system. It was found that the matrix had little effect on analyte signal intensities when small volumes (0.1 to 1 μL) of crude samples or 1 : 10 diluted samples were injected into the LC-ESI-MS/MS system. The standard addition method was also investigated for quantitative assessment but proved unsuccessful, possibly due to poor ionisation of the electrospray interface at high analyte concentrations. Therefore a method using external calibration was used for semi-quantitative determination of the analytes in decomposition fluid. The LC-ESI-MS/MS method enabled identification of all target compounds as being present. In addition a 14-day cyclic trend in the concentrations of two amino acids, phenylalanine and tryptophan, was tentatively established. General increasing trends with respect to time and temperature were also observed for putrescine and indole.

Determination of aminoglycoside residues by liquid chromatography and tandem mass spectrometry in a variety of matrices

A quantitative LC–MS/MS method was developed for the determination of 13 commonly used aminoglycoside antibiotics in meat (pork muscle, fish, and veal livers and kidneys). The proposed method is sufficiently sensitive and highly selective. Unlike other previously reported methods, it uses a simple clean-up procedure based on a strong cation-exchange solid-phase cartridge that permits high sample extract loading volumes. A unique elution regime based on a volatile buffer at intermediately high pH value in combination with an organic solvent provides quantitative elution of the various aminoglycosides. This methodology ensured that neither a breakthrough of weakly retained aminoglycosides (e.g. spectinomycin) nor the incomplete elution of strongly retained analytes (e.g. neo- and gentamycin) is observed. The single-step clean-up is fast and produces clean extracts that minimize matrix-related signal suppression in the electrospray interface.