Ultra-performance liquid chromatography coupled to quadrupole-orthogonal time-of-flight mass spectrometry

Ultra-performance liquid chromatography (UPLC) utilizes sub-2 microm particles with high linear solvent velocities to effect dramatic increases in resolution, sensitivity and speed of analysis. The reduction in particle size to below 2 microm requires instrumentation that can operate at pressures in the 6000-15,000 psi range. The typical peak widths generated by the UPLC system are in the order of 1-2 s for a 10-min separation. In the present work this technology has been applied to the study of in vivo drug metabolism, in particular the analysis of drug metabolites in bile. The reduction in peak width significantly increases analytical sensitivity by three- to five-fold, and the reduction in peak width, and concomitant increase in peak capacity, significantly reduces spectral overlap resulting in superior spectral quality in both MS and MS/MS modes. The application of UPLC/MS resulted in the detection of additional drug metabolites, superior separation and improved spectral quality.     

Quantitative multiresidue method for about 100 veterinary drugs in different meat matrices by sub 2-microm particulate high-performance liquid chromatography coupled to time of flight mass spectrometry

A quantitative multiresidue method covering more than 100 veterinary drugs, belonging to different drug families, has been developed. The proposed approach uses an liquid-liquid-solid extraction technique (bi-polarity extraction) which is capable in recovering polar, medium polar and apolar compounds. A thorough generic reversed phase solid-phase extraction (SPE) clean-up removes interfering proteins and provides clean and stable extracts. Dedicated rinsing steps are proposed to reduce analyte adsorption on glass walls and on precipitating proteins. The resulting extract is analyzed by ultra-performance liquid chromatography (UPLC) coupled to time of flight mass spectrometry (TOF). The method was validated according to the Commission Decision 2002/657/EEC. Validation coved the relevant meat matrices (muscle, kidney and liver).     

Ultra-performance liquid chromatography-tandem mass spectrometry: a novel challenge in multiresidue pesticide analysis in food

Potential of ultra-performance liquid chromatography (UPLC) separation strategy coupled with tandem (in space) mass spectrometric detection (MS/MS) in multiresidue pesticide analysis was critically assessed. Performance parameters such as number of theoretical plates, height of theoretical plate, peak symmetry and peak capacity were measured/calculated on the basis of data generated by analysis of apple extracts containing 17 (semi)polar pesticides representing various classes of active ingredients of widely used crop protective preparations. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) procedure provided improved chromatographic parameters resulting in significantly increased sample throughput including lower solvent consumption and lower limits of quantitation (LOQs) for most of target analytes compared to common method employing conventional high-performance liquid chromatography (HPLC) separation.     

Fast screening and quantitation of microcystins in microalgae dietary supplement products and water by liquid chromatography coupled to time of flight mass spectrometry

Cyanobacteria, commonly called "blue-green algae", may accumulate in surface water supplies as "blooms" and may concentrate on the surface as blue-green "scums". Some species of cyanobacteria produce toxins and are of relevance to water supplies and to microalgae dietary supplements. To ensure the safety of drinking water and blue-green algae products, analyses are the only way to determine the presence or absence of toxins. This paper shows the use of ultra performance liquid chromatography (UPLC) coupled to orthogonal acceleration time of flight (TOF) mass spectrometry for the detection and quantitation of microcystins. The method presented is very sensitive, simple, fast, robust and did not require fastidious clean-up step. Limits of detection of 0.1 microg L(-1) in water and 0.1-0.2 microg g(-1) in microalgae samples were achieved. Method performances were satisfactory and appropriate for monitoring of water and dietary supplements. The method was applied in routine to samples taken from Swiss market or buy on internet website. Among 19 samples, six showed the presence of microcystins LR and LA at harmful levels.     

Ultra-performance liquid chromatography coupled to linear ion trap mass spectrometry for the identification of drug metabolites in biological samples

The coupling of ultra-performance liquid chromatography, operating at elevated pressures, to a linear ion trap mass spectrometer provides a high-performance system suitable for drug metabolite characterisation. This system demonstrates improved chromatographic efficiency and sensitivity and at the same time provides diagnostic MSn data often critical for metabolite structural assignment. The linear ion trap was capable of dealing with the high chromatographic efficiencies and hence narrow peak widths associated with 1.7 microm particle-packed column separations. Polarity switching and data-dependent MSn data were generated with ease, and applied to the identification of metabolites found in human plasma.     

Multi-residue screening of veterinary drugs in egg,fish and meat using high-resolution liquid chromatography accurate mass time-of-flight mass spectrometry

The last 2 years multi-compound methods are gaining ground as screening methods. In this study a high-resolution liquid chromatography combined with time-of-flight mass spectrometry (HRLC–ToF-MS) is tested for the screening of about 100 veterinary drugs in three matrices, meat, fish and egg. While the results are satisfactory for 70–90% of the veterinary drugs, a more efficient sample preparation or extract purification is required for quantitative analysis of all analytes in more difficult matrices like egg. The average mass measurement error of the ToF-MS for the veterinary drugs spiked at concentrations ranging from 4 to 400 μg/kg, is 3.0 ppm (median 2.5 ppm) with little difference between the three matrices, but slightly decreases with increasing concentration. The SigmaFit value, a new feature for isotope pattern matching, also decreases with increasing concentration and, in addition, shows an increase with increasing matrix complexity. While the average SigmaFit value is 0.04, the median is 0.01 indicating some high individual deviations. As with the mass measurement error, the highest deviations are found in those regions of the chromatogram where most compounds elute from the column, be it analytes or matrix compounds. The median repeatability of the method ranges from 8% to 15%, decreasing with increasing concentration, while the median reproducibility ranges from 15% to 20% with little difference between matrices and concentrations. The median accuracy is in between 70% and 100% with a few compounds showing higher values due to matrix interference. The squared regression coefficient is >0.99 for 92% of the compounds showing a good overall linearity for most compounds. The detection capability, CCβ, is within 2 times the associated validation level for >90% of the compounds studied. By changing a few conditions in the analyses protocol and analysing a number of blank samples, it was determined that the method is robust as well as specific. Finally, an alternative validation strategy is proposed and tested for screening methods. While the results calculated for repeatability, within-lab reproducibility and CCβ show a good comparison for the matrices meat and fish, and a reasonable comparison for the matrix egg, only 27 analyses are required to obtain these results versus 63 analysis in the traditional, 2002/657/EC, approach. This alternative is suggested as a cost-effective validation procedure for screening methods.     

Metabonomics: the use of electrospray mass spectrometry coupled to reversed-phase liquid chromatography shows potential for the screening of rat urine in drug development

The application of liquid chromatography/mass spectrometry (LC/MS) followed by principal components analysis (PCA) has been successfully applied to the screening of rat urine following the administration of three candidate pharmaceuticals. With this methodology it was possible to differentiate the control samples from the dosed samples and to identify the components of the mass spectrum responsible for the separation. These data clearly show that LC/MS is a viable alternative, or complementary, technique to proton NMR for metabonomics applications in drug discovery and development.     

Development of a broad toxicological screening technique for urine using ultra-performance liquid chromatography and time-of-flight mass spectrometry

Withdrawal of the support for the REMEDi HS drug profiling system has necessitated its replacement within our laboratories with an alternative broad toxicological screening technique. To this end, a novel method, based on ultra-performance liquid chromatography (UPLC) and time-of-flight (TOF) mass spectrometry, was developed for the routine analysis of urine samples. Identification was achieved by comparison of acquired data to libraries containing more than 300 common drugs and metabolites, and was based on a combination of retention time, exact mass and fragmentation patterns. Validation data for the method is presented and comprised an evaluation of the following parameters: precision; transferability of the methodology between the six collaborating laboratories; specificity; extraction recovery and stability of processed samples; matrix effects and sensitivity.This paper presents the benefits of supplementary fragmentation data with particular regard to increasing specificity and confidence of identification and its usefulness with overdosed samples. The utility of the method was assessed by the parallel analysis of 30 authentic urine samples using the REMEDi HS and UPLC-TOF. The latter provided enhanced detection, leading to the identification of twice as many drugs. Furthermore it did not miss any compounds that were identified by REMEDi HS. The UPLC-TOF findings were further verified by a combination of data from three other conventional screening techniques, i.e., GC-MS, HPLC-DAD and UPLC-MS/MS.     

A high-sensitivity ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry (UHPLC-HR-TOFMS) method for screening synthetic cannabinoids and other drugs of abuse in urine

The continuing emergence of designer drugs imposes high demands on the scope and sensitivity of toxicological drug screening procedures. An ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry (UHPLC-HR-TOFMS) method was developed for screening and simultaneous confirmation of both designer drugs and other drugs of abuse in urine samples in a single run. The method covered selected synthetic cannabinoids and cathinones, amphetamines, natural cannabinoids, opioids, cocaine and other important drugs of abuse, together with their main urinary metabolites. The database consisted of 277 compounds with molecular formula and exact monoisotopic mass; retention time was included for 192 compounds, and primary and secondary qualifier ion exact mass for 191 and 95 compounds, respectively. Following a solid-phase extraction, separation was performed by UHPLC and mass analysis by HR-TOFMS. MS, and broad-band collision-induced dissociation data were acquired at m/z range 50–700. Compound identification was based on a reverse database search with acceptance criteria for retention time, precursor ion mass accuracy, isotopic pattern and abundance of qualifier ions. Mass resolving power in spiked urine samples was on average FWHM 23,500 and mass accuracy 0.3 mDa. The mean and median cut-off concentrations determined for 75 compounds were 4.2 and 1 ng/mL, respectively. The range of cut-off concentrations for synthetic cannabinoids was 0.2–60 ng/mL and for cathinones 0.7–15 ng/mL. The method proved to combine high sensitivity and a wide scope in a manner not previously reported in drugs of abuse screening. The method’s feasibility was demonstrated with 50 authentic urine samples.

Fully automated screening of veterinary drugs in milk by turbulent flow chromatography and tandem mass spectrometry

There is an increasing interest in screening methods for quick and sensitive analysis of various classes of veterinary drugs with limited sample pre-treatment. Turbulent flow chromatography in combination with tandem mass spectrometry has been applied for the first time as an efficient screening method in routine analysis of milk samples. Eight veterinary drugs, belonging to seven different classes were selected for this study. After developing and optimising the method, parameters such as linearity, repeatability, matrix effects and carry-over were studied. The screening method was then tested in the routine analysis of 12 raw milk samples. Even without internal standards, the linearity of the method was found to be good in the concentration range of 50 to 500 μg/L. Regarding repeatability, RSDs below 12% were obtained for all analytes, with only a few exceptions. The limits of detection were between 0.1 and 5.2 μg/L, far below the maximum residue levels for milk set by the EU regulations. While matrix effects—ion suppression or enhancement—are obtained for all the analytes the method has proved to be useful for screening purposes because of its sensitivity, linearity and repeatability. Furthermore, when performing the routine analysis of the raw milk samples, no false positive or negative results were obtained.     

High-performance liquid chromatography coupled to mass spectrometry for studying new pharmaceutical entities

The review covers the aspects of the use of high-performance liquid chromatography coupled to mass spectrometry for studying new pharmaceutical entities. Attention is paid mainly to pharmacokinetic investigations. Different methods of sample preparation and approaches to minimize the duration of the analysis are discussed.     

Gas and liquid chromatography coupled to tandem mass spectrometry for the multiresidue analysis of β-agonists in biological matrices

β-Agonists are substances used in veterinary and human medicine for the treatment of pulmonary disorders. They have found a use as growth promoters to improve meat-to-fat ratios in cattle but they are not authorized for use in the European Union. Due to their presence in trace levels (usually less than 1 μg/kg), to the diversity of the illegally used compounds and to the complexity of the biological matrices analysed, the detection of these residues requires a very sensitive and specific method of determination. This work describes the strategy of analysis we developed for five β-agonists in urine and liver. The combination of improved solid- or liquid-phase extraction methods and LC or GC-MS-MS (in the multiple reaction monitoring mode) has shown to provide a system suitable for the control of these substances. The efficiency of extraction and the sensitivity and selectivity allow this multiresidue detection down to, and below, the UK regulatory level of 0.5 μg/kg. Moreover, the use of LC removes the need for the derivatisation step (cyclic methylboronate derivatives) which is required prior to GC-MS-MS analysis.     

Wide-scope analysis of veterinary drug and pesticide residues in animal feed by liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry

A fast and generic method has been developed for the simultaneous monitoring of >250 pesticides and veterinary drugs (VDs) in animal feed. A ‘dilute-and-shoot’ extraction with water and acetonitrile (1 % formic acid) followed by a clean-up step with Florisil cartridges was applied. The extracts were analysed by ultra-high performance liquid chromatography coupled to hybrid analyser quadrupole-time-of-flight mass spectrometry using both positive and negative electrospray ionisation. The detection of the residues was accomplished by retention time and accurate mass using an in-house database. The identification of the detected compounds was carried out by searching of fragment ions for each compound and isotopic pattern. The optimised method was validated and recoveries ranged from 60 % to 120 % at three concentrations (10, 50 and 100 μg kg^?1) for 30 %, 68 % and 80 % of compounds, respectively, included in the database (364) in chicken feed. Document SANCO 12495/2011 and Directive 2002/657/CE were used as guidelines for method validation. Intra-day and inter-day precisions, expressed as relative standard deviations, were lower than 20 % for more than 90 % of compounds. The limits of quantification ranged from 4 to 200 μg kg^?1 for most analytes, which are sufficient to verify compliance of products with legal tolerances. The applicability of the procedure was further tested on different types of feed (chicken, hen, rabbit and horse feed), evaluating recoveries and repeatability. Finally, the method was applied to the analysis of 18 feed samples, detecting some VDs (sulfadiazine, trimethoprim, robenidin and monensin Na) and only one pesticide (chlorpyrifos).     

Validation of a screening method based on liquid chromatography coupled to high-resolution mass spectrometry for analysis of perfluoroalkylated substances in biota

A screening method for analysis of perfluoroalkylated substances (PFAS) in biota samples has been developed and validated using liver samples from polar cod (Boreogadus saida) and glaucous gull (Larus hyperboreus). The method was based on extraction of target compounds from homogenised samples into the solvent mixture used as mobile phase in high-performance liquid chromatography (HPLC), i.e. methanol/water (50:50; 2 mM ammonium acetate). The extract was filtered and directly injected into a HPLC/time-of-flight mass spectrometry (TOF-MS) system. Quantification was performed using 7H-perfluoroheptanoic acid as internal standard and a calibration standard solution dissolved in sample extract for each matrix type (matrix-matched calibration standard). The method is very time and cost efficient. Except for long-chain compounds and perfluorooctane sulfonamide (which cannot be covered by this method), recoveries were between 60% and 115% and method detection limits were in the range 0.04-1.3 ng/g wet weight. Blank values could be neglected with the exception of perfluorooctane sulfonate (PFOS), perfluorohexanoic acid (PFHxA) and perfluorooctanoic acid (PFOA). One of the major challenges in PFAS analysis is ionisation disturbance by co-eluting matrix in the ion source of the mass spectrometer. Both matrix and analyte specific signal enhancement and suppression was observed and quantified. Repeated extractions (n = 3) gave relative standard deviations (RSD) <35% for all PFAS. Accuracy was examined by comparing the screening method to the generally applied ion pair extraction (IPE) method. PFAS concentration values of a glaucous gull liver sample deviated by less than 30% for the two methods, provided that matrix-matched calibration standards were employed in both methods.     

Ultra-performance liquid chromatography/tandem mass spectrometry method for the determination of lercanidipine in human plasma

A simple, sensitive and rapid ultra-performance liquid chromatography/positive electrospray ionization tandem mass spectrometry (UPLC/ESI-MS/MS) method has been developed and validated for the determination of lercanidipine in human plasma. Lercanidipine and the internal standard, nicardipine, were extracted from plasma by liquid-liquid extraction using tert-butyl methyl ether as the extraction solvent. UPLC analysis was performed isocratically on an AcQuity UPLC BEH C18 analytical column (2.1 x 50.0 mm i.d., particle size 1.7 microm). The mobile phase consisted of 70% acetonitrile in water containing 0.2% v/v formic acid and pumped at a flow rate of 0.30 mL/min. ESI in positive ion mode, with multiple reaction monitoring (MRM), was chosen for the detection of the analytes. The assay was linear over a concentration range of 0.05-30 ng/mL for lercanidipine with a limit of quantitation of 0.05 ng/mL. Quality control samples (0.05, 0.15, 15 and 25 ng/mL) in five replicates from five of analytical runs demonstrated intra-assay precision (% CV < or =7.3%), inter-assay precision (% CV < or =6.1%) and an overall accuracy (% relative error) of less than 6.2%. A run time of less than 1.0 min for each sample made it possible to analyze a large number of human plasma samples per day. The method can be used to quantify lercanidipine in human plasma covering a variety of pharmacokinetic or bioequivalence studies.     

Development of a simple liquid chromatography-tandem mass spectrometry method for multiresidue determination of antifungal drugs in chicken tissues

A method involving LC coupled with MS/MS (LC/MS/MS) was designed for simultaneous quantification of 10 antifungal drugs (voriconazole, griseofulvin, clotrimazole, bifonazole, econazole, ketoconazole, itraconazole, miconazole, terconazole, and fluconazole) in the liver and muscles of chickens. Homogenized tissue samples were extracted with acetonitrile and subsequently underwent freezing-delipidation. A Waters Acquity Ultra Performance LC BEH C18 column was used to separate the analytes, coupled with MS/MS using an electrospray ionization source. The accuracy of the method was confirmed with a mean recovery of 71-121%, and acceptable coefficients of variation (4-23%, n = 6). The detection capability of these compounds in two different matrixes was 0.50-2.82 microg/kg. This method can be applied for the screening and confirmation of target antifungal drugs in chicken tissues.     

Determination of vitamin B5 in human urine by high-performance liquid chromatography coupled with mass spectrometry

In the present work, we have developed a simple and rapid liquid chromatography/mass spectrometry (LC/MS) method for the identification and quantification of vitamin B5 in human urine. Urine was spiked with vitamin B5 internal standard, hopantenic acid (HOPA), and then diluted with the LC mobile phase prior to its analysis by LC/MS. The quantification was performed in single ion monitoring mode. The calibration curve was linear (r2 = 0.999) between 0.25 to 10 microg/mL. With a limit of detection of 0.1 microg/mL the method was sensitive enough to determine low levels of vitamin B5 in urine. The overall quantitative efficiency of the method was evaluated by spiking urine samples with four different concentrations of vitamin B5; the intra-assay coefficient of variation was below 5% and the recoveries were between 96 to 108%. The results of the present study show that the proposed method is selective and sensitive enough for the quantification of vitamin B5 in urine.     

Quantitative trace analysis of eight chloramphenicol isomers in urine by chiral liquid chromatography coupled to tandem mass spectrometry

Chloramphenicol is a broad-spectrum antibiotic with, apart from its human medicinal use, veterinary abuse in all major food-producing animals. Chloramphenicol occurs in four stereoisomers (all para-nitro substituted) and furthermore four meta-nitro analogs of chloramphenicol exist. In this paper these are referred to as eight chloramphenicol isomers. According to EU regulations an analytical method should be able to discriminate the analyte from interfering substances that might be present in the sample, including isomers. For the first time a quantitative method for the analysis of trace levels of eight chloramphenicol isomers in urine by chiral liquid chromatography in combination with tandem mass spectrometric detection is reported. The separation of the isomers on the analytical column, the clean-up of urine and the selectivity of the monitored product ions turned out to be critical parameters. To obtain reproducible retention isocratic elution on a chiral AGP column was applied. For urine samples matrix compounds present in the final extract caused decreased retention of the isomers on the chiral stationary phase and a lack of chromatographic resolution. Therefore an extended clean-up procedure that combines solid phase extraction and liquid-liquid extraction had to be developed. The final method was fully validated and showed satisfactory performance for all isomers with decision limits (CCα) ranging from 0.005 to 0.03 μg L(-1) and within-laboratory reproducibility of all isomers below 20% at the minimum required performance limit level of 0.3 μg L(-1).     

Isotopic pattern and accurate mass determination in urine drug screening by liquid chromatography/time-of-flight mass spectrometry

An efficient method was developed for toxicological drug screening in urine by liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry. The method relies on a large target database of exact monoisotopic masses representing the elemental formulae of reference drugs and their metabolites. Mass spectral identification is based on matching measured accurate mass and isotopic pattern (SigmaFit) of a sample component with those in the database. Data post-processing software was developed for automated reporting of findings in an easily interpretable form. The mean and median of SigmaFit for true-positive findings were 0.0066 and 0.0051, respectively. The mean and median of mass error absolute values for true-positive findings were 2.51 and 2.17 ppm, respectively, corresponding to 0.65 and 0.60 mTh. For routine screening practice, a SigmaFit tolerance of 0.03 and a mass tolerance of 10 ppm were chosen. Ion abundance differences from urine extracts did not affect the accuracy of the automatically acquired SigmaFit or mass values. The results show that isotopic pattern matching by SigmaFit is a powerful means of identification in addition to accurate mass measurement.