Oscar J. Pozo

Rapid direct determination of pesticides and metabolites in environmental water samples at sub-μg/l level by on-line solid-phase extraction-liquid chromatography-electrospray tandem mass spectrometry

A very rapid, multi-residual, sensitive and specific procedure for determining 35 pesticides in environmental ground and surface water in proposed. It is based on the use of solid-phase extraction (SPE) combined on-line with liquid chromatography (LC) electrospray (ESI) tandem mass spectrometry (MS-MS). Simultaneous target analysis of 29 pesticides (1 fungicide, 16 insecticides, 10 herbicides and 2 acaricides) and 6 metabolites with positive or negative ionization was reached by the direct injection of only 1.3 ml of filtered water sample, with a total analysis time of 18 min. The SPE-LC-MS-MS method was validated, obtaining good results for all compounds at 0.5 and 0.1 microg/l. Most of them could be correctly quantified at a concentration level as low as 25 ng/l. Efficiency and applicability of this method was evaluated by the analysis of several samples included in a monitoring program.

Efficient approach for the comprehensive detection of unknown anabolic steroids and metabolites in human urine by liquid chromatography-electrospray-tandem mass spectrometry

The detection of new anabolic steroid metabolites and new designer steroids in urine is a challenge in doping analysis. An approach based on precursor ion scanning for the detection of unknown anabolic steroids and metabolites is proposed. The study of the MS/MS spectra of selected anabolic steroids revealed different fragmentation pathways at low and medium collision energy depending on the steroid structure. However, after analysis at high collision energy three common ions at m/z 105, m/z 91, and m/z 77 were found for all studied anabolic steroids. These ions can be explained by the fragmentation of the steroid structure and corresponded to the methyl tropylium, tropylium, and phenyl ions, respectively. Because of the theoretical low specificity of these ions, the simultaneous presence of all of them was used as a starting point to consider a substance as a possible anabolic steroid. Hence, the developed approach is based on the simultaneous acquisition of the precursor ion scan of m/z 105, 91, and 77. The specificity of this approach has been checked by the injection of several doping agents including beta-agonists, corticosteroids, beta-blockers, and diuretics. In general, only compounds with a steroidal structure showed a signal at all three selected m/z values although some exceptions have been found. The applicability of the method was tested for three different scenarios: the detection of steroid metabolites, the detection of unknown steroids, and the analysis of prohormones. In metabolic studies, several recently reported fluoxymesterone metabolites were also found using this method. For detection of unknown steroids, some negative urine samples were spiked with the designer steroid THG and 33 other anabolic steroids and treated as blind samples. Finally, the applicability of the developed approach for the analysis of dietary supplements was checked by the analysis of a prohormone where several impurities and/or degradation products were found.

Collision-induced dissociation of 3-keto anabolic steroids and related compounds after electrospray ionization. Considerations for structural elucidation

The collision-induced dissociation of forty-one 3-keto anabolic steroids and related compounds has been studied using both triple quadrupole (QqQ) and hybrid quadrupole-time of flight (QTOF) instruments. Due to the complexity of the product ion spectra of these analytes, which generate a large number of ions, only two specific regions were studied in depth: the product ions near the precursor ion (m/z > or =M-100) and the most abundant product ions at a collision energy of 30 eV. Accurate mass measurements were used in order to obtain an unequivocal assignment of the empirical formula and the origin of each selected product ion. Analytes have been divided into eight groups according to the number and position of double bonds and the presence of functional groups such as hydroxyl- or nitrogen-containing rings. A correlation between the steroid structure and the product ions obtained has been postulated. The application of these correlations can be useful in the elucidation of feasible structures for unknown steroids and/or their metabolites.

Direct quantification of steroid glucuronides in human urine by liquid chromatography-electrospray tandem mass spectrometry.

A method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the direct quantification of glucuronides of testosterone (TG), epitestosterone (EPG), androsterone (AG) and etiocholanolone (ETG) has been developed. The method allowed for the direct determination of these analytes avoiding hydrolysis and derivatization, which are usual steps in commonly used methods based on gas chromatography-mass spectrometry (GC-MS). The electrospray ionization and the product ion spectra of the glucuronides have been studied in order to obtain the most specific transitions. The use of the selected transitions is necessary for the determination of the analytes at low ng/ml concentration levels. Two different approaches have been tested for sample preparation: direct injection after filtration and acidic liquid-liquid extraction (LLE) with ethyl acetate. Both approaches have been validated obtaining satisfactory values for accuracy and precision with limits of detection lower than 1 ng/ml for TG and EPG. Ion suppression was more pronounced after LLE probably due to the concentration of interferences from acidic urine. The applicability of the method has been checked by the analysis of 40 urine samples. The results were compared with those obtained with the common GC-MS method. Results have shown a good correlation between both methods with correlation coefficients higher than 0.97. A slope close to 1 was obtained for all analytes except for AG possibly due to losses during the extraction process prior to GC-MS.

Study of matrix effects on the direct trace analysis of acidic pesticides in water using various liquid chromatographic modes coupled to tandem mass spectrometric detection.

This study investigated the effects of matrix interferences on the analytical performance of a triple quadrupole mass spectrometric (MS-MS) detector coupled to various reversed-phase liquid chromatographic (LC) modes for the on-line determination of various types of acidic herbicides in water using external calibration for quantification of the analytes tested at a level of 0.4 microg/l. The LC modes included (i) a single-column configuration (LC), (ii) precolumn switching (PC-LC) and (iii) coupled-column LC (LC-LC). As regards detection, electrospray (ESI) and atmospheric pressure chemical ionization (APCI) in both positive (PI) and negative (NI) ionization modes were examined. Salinity and dissolved organic carbon (DOC) were selected as interferences to study matrix effects in this type of analysis. Therefore, Milli-Q and tap water samples both fortified with 12 mg/l DOC and spiked with sulfometuron-methyl, bentazone, bromoxynil, 2-methyl-4-chlorophenoxyacetic acid, and 2-methyl-4-chlorophenoxypropionic acid at a level of about 0.4 microg/l were analyzed with the various LC-MS approaches. Direct sample injection was performed with volumes of 0.25 ml or 2.0 ml on a column of 2.1 mm I.D. or 4.6 mm I.D. for the ESI and APCI modes, respectively. The recovery data were used to compare and evaluate the analytical performance of the various LC approaches. As regards matrix effects, the salinity provided a dramatic decrease in response for early eluting analytes (k value of about 1) when using the LC mode. Both PC-LC and LC-LC efficiently eliminated this problem. The high DOC content hardly effected the responses of analytes in the ESI mode, while in most cases the responses increased when using APCI-MS-MS detection. Of all the tested configurations, LC-LC-ESI-MS-MS with the column combination Discovery C18/ABZ+ was the most favorable as regards elimination of matrix effects and provided reliable quantification of all compounds using external calibration at the tested low level. The major observed effects were verified with statistical evaluation of the data employing backwards ordinary least-square regression. All tested column-switching modes hyphenated to ESI- or APCI-MS-MS allowed the on-line multi-residue analysis of acidic pesticides in the reference water down to a level of 0.1 microg/l in less than 10 min, emphasizing the feasibility of such an approach in this field of analysis.

Determination of the herbicide 4-chloro-2-methylphenoxyacetic acid and its main metabolite, 4-chloro-2-methylphenol in water and soil by liquid chromatography–electrospray tandem mass spectrometry

A rapid and sensitive LC-electrospray tandem mass spectrometry method has been developed for the quantitation of 4-chloro-2-methylphenoxyacetic acid (MCPA) and 4-chloro-2-methylphenol in both water and soil samples. Soil samples were extracted in alkaline media and cleaned-up by solid-phase extraction with C18 cartridges before LC-MS analysis. The selectivity and sensitivity offered by the triple quadrupole allowed the direct injection of the water samples rendering a sample throughput of around 100 samples per day, without any sample pretreatment, rendering for MCPA a limit of detection of 40 ng/l. In order to increase the method sensitivity, mainly for metabolite, a previous solid-phase extraction step was also performed. The method was validated by means of recovery experiments using fortified water and soil samples, obtaining satisfactory recoveries for both compounds in water and for MCPA in soil. The validated procedures can be used for the specific monitoring of residues of MCPA and its main metabolite in environmental samples, as ground and surface waters and soils, providing more selectivity and sensitivity than the current UV-based methodology. Besides, sample manipulation is greatly reduced in comparison to other GC-MS based methods which require a previous derivatization.

Quantification, confirmation and screening capability of UHPLC coupled to triple quadrupole and hybrid quadrupole time-of-flight mass spectrometry in pesticide residue analysis.

The potential of three mass spectrometry (MS) analyzers (triple quadrupole, QqQ; time of flight, TOF; and quadrupole time of flight, QTOF) has been investigated and compared for quantification, confirmation and screening purposes in pesticide residue analysis of fruit and vegetable samples. For this purpose, analytical methodology for multiresidue determination of 11 pesticides, taken as a model, has been developed and validated in nine food matrices for the three mass analyzers coupled to ultra high pressure liquid chromatography. In all cases, limits of quantification around 0.01 mg/kg were reached, fulfilling the most restrictive case of baby-food analysis. Regarding absolute sensitivity, the lower limits of detection were obtained, as expected, for QqQ (100 fg), whereas slightly higher limits (300 fg) were obtained for both TOF and QTOF. Confirmative capacity of each analyzer was studied for each analyte based on the identification points (IPs) criterion, useful for a comprehensive comparison. QTOF mass analyzer showed the highest confirmatory capacity, although QqQ normally led to sufficient number of IPs, even at lower concentration levels. The potential of TOF MS was also investigated for screening purposes. To this aim, around 50 commercial fruits and vegetables samples were analyzed, searching for more than 400 pesticides. TOF MS proved to be an attractive analytical tool for rapid detection and reliable identification of a large number of pesticides thanks to the full spectrum acquisition at accurate mass with satisfactory sensitivity. This process is readily boosted when combined with specialized software packages, together with theoretical exact mass databases. Several pesticides (e.g. carbendazim in citrus and indoxacarb in grape) were detected in the samples. Further unequivocal confirmation of the identity was performed using reference standards and/or QTOF MS/MS experiments.

Determination of abamectin and azadirachtin residues in orange samples by liquid chromatography-electrospray tandem mass spectrometry

A rapid and sensitive LC-ESI-MS-MS method has been developed for the determination of azadirachtin and abamectin residues in orange samples. Samples were extracted with acetonitrile, in a high-speed blender. After the addition of sodium acetate, an aliquot of extract was directly injected into the LC-ESI-MS-MS system. The highest sensitivity of the method was achieved under MS-MS conditions using [M+Na]+ adducts as precursor ions. Recoveries for both compoundsfrom spiked orange samples at 0.01 and 0.1 mg/kg were above 80%, with good repeatability (<10%). Method detection limits achieved (<0.007 mg/kg) were adequate for the determination of these pesticides in this kind of sample from the regulatory point of view. The importance of the solvent used for extraction, as well as the addition of sodium acetate to the extracts and the selection of adequate chromatographic conditions are discussed.

Use of LC-MS/MS for the Open Detection of Steroid Metabolites Conjugated with Glucuronic Acid

In humans, conjugation with glucuronic acid is the most important phase II metabolic reaction of steroidal compounds. Glucuronoconjugated metabolites have been conventionally studied by using β-glucuronidase enzymes to release the phase I metabolites. It is well-known that hydrolysis with β-glucuronidase presents some limitations that may result in the underestimation of some conjugates. The aim of the present work was to develop and to evaluate liquid chromatography-tandem mass spectrometry (LC-MS/MS) scan methods for the open detection of steroid glucuronides in urine samples. The mass spectrometric behavior of thirteen representative steroid glucuronides, used as model compounds, was studied. Characteristic ionization and collision induced dissociation behaviors were observed depending on the steroid glucuronide structure. Neutral loss (NL of 176, 194, 211, and 229 Da) and precursor ion (PI of m/z 141, 159, and 177, in positive mode and m/z 75, 85, and 113, in negative mode) scan methods were evaluated. The NL scan method was chosen for the open detection of glucuronoconjugated steroids due to its sensitivity and the structural information provided by this method. The application of the NL scan method to urine samples collected after testosterone (T) undecanoate administration revealed the presence of two T metabolites which remain conjugated as glucuronides after an enzymatic hydrolysis of the urine. 3α,6β-Dihydroxy-5α-androstan-17-one (6β-hydroxyandrosterone) glucuronide and 3α,6β-dihydroxy-5β-androstan-17-one (6β-hydroxyetiocholanolone) glucuronide were established as the structures for these metabolites, by comparing the structure of the steroids released after chemical hydrolysis with reference materials. An increase of 50-300-fold of these metabolites after oral administration of T undecanoate was observed, proving that their determination can be useful in the doping control field. Moreover, these results exemplify that significant information might be missed, unless direct methods for the determination of steroid glucuronides are employed.

Direct determination of chlorpyrifos and its main metabolite 3,5,6‐trichloro‐2‐pyridinol in human serum and urine by coupled‐column liquid chromatography/electrospray‐tandem mass spectrometry

A rapid and sensitive automated coupled-column liquid chromatography/electrospray tandem mass spectrometry (LC/LC/ES-MS/MS) method has been developed for the quantitation of chlorpyrifos and 3,5,6-trichloro-2-pyridinol (TCP) in both human serum and urine. Human serum was first protein precipitated with acetonitrile, while urine was directly injected into the coupled-column system. A 10 microL aliquot was then analyzed using as first separation column a Discovery C18 5 microm 50 x 2.1 mm; the fraction containing the analyte was transferred on-line to the second column consisting of a ABZ+ 5 microm 100 x 2.1 mm, which was connected to the electrospray source (Z-spray) of a Quattro LC triple-quadrupole instrument. Chlorpyrifos was detected in positive ion mode using four multi reaction monitoring (MRM) transitions while TCP was measured in negative ion mode using three pseudo-MRM transitions. The clean-up performed by the coupled-column approach avoids the use of an internal standard for the correct quantitation of both analytes, and the highly automated procedure renders a sample throughput of more than 100 samples per day. Both compounds can be determined using the same set-up, the only difference in the procedure being the composition of the first mobile phase. The method has proved to be fast, reliable and sensitive, yielding calibration curves for both analytes with correlation coefficients greater than 0.9995. The repeatability and reproducibility at 5 and 50 ng/mL was lower than 8%. The accuracy and precision were evaluated by means of recovery experiments from fortified serum (5-50 ng/mL) and urine (1-10 ng/mL) samples, obtaining satisfactory recoveries for both compounds (87-113% in serum, and 98-109% in urine), with coefficients of variation (CVs) less than 10%. The detection limits were similar for chlorpyrifos and metabolite: 1.5 ng/mL in serum, and 0.5 ng/mL in urine, where no sample handling took place. The validated procedures provide excellent tools for the specific assessment of occupational exposure to the organophosphorus pesticide chlorpyrifos, throughout the analysis of both human serum and urine, and it is more selective and sensitive than the current assay based on the measurement of the decrease in the cholinesterase activity.