Electrospray and chemical ionization mass spectrometry of di-n-butyl sulfate. Unimolecular chemistry of its protonated form and quantification method by liquid chromatography/electrospray ionization tandem mass spectrometry

Di-n-butyl sulfate (DNBS) has been studied by electrospray (ESI) and chemical (CI) ionization mass spectrometry. The use of methanol as solvent in electrospray ionization allows observation of relatively abundant [DNBS + CH(3)OH + H](+) ions (m/z 243) which upon collision dissociate to [DNBS + H](+) ions (m/z 211). In both ESI and CI experiments, it is found that [DNBS + H](+) ions lead to m/z 113 daughter ions. The composition of this m/z 113 fragment ion and its mechanism of formation have been established by high resolution measurements and CID-MIKE experiments. An 'internal substitution' reaction involving an ion-neutral intermediate is proposed to explain the formation of a [C(8)H(17)](+) ion (m/z 113) by loss of a H(2)SO(4) molecule. Finally, a LC/ESI-MS/MS quantification method is proposed in which a detection limit of di-n-butyl sulfate in the ppm range is obtained. It is suggested that the quantification method might be extended to higher dialkyl sulfates. Copyright 2000 John Wiley & Sons, Ltd.     

Determination of Tripamide in human urine by high-performance liquid chromatography and high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

Tripamide is a drug widely used in clinical practice for the treatment of hypertension and edema. This work evaluated a screening method for Tripamide and its urinary metabolites in human urine, using high-performance liquid chromatography diode-array detection (HPLC/DAD). Identification of these metabolites was investigated by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) after dosing with 15 mg Tripamide. Acid hydrolysis showed that Tripamide is conjugated in the body. Two suspected metabolites were detected by HPLC/DAD. HPLC/ESI-MS/MS analysis suggested that these metabolites were probably hydroxylated together with loss of the -NH(2) group and dehydrogenation. These results will be useful in confirmation methods for Tripamide in doping control.     

Derivatization reagents in liquid chromatography/electrospray ionization tandem mass spectrometry

Liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) is one of the most prominent analytical techniques owing to its inherent selectivity and sensitivity. In LC/ESI-MS/MS, chemical derivatization is often used to enhance the detection sensitivity. Derivatization improves the chromatographic separation, and enhances the mass spectrometric ionization efficiency and MS/MS detectability. In this review, an overview of the derivatization reagents which have been applied to LC/ESI-MS/MS is presented, focusing on the applications to low molecular weight compounds.     

Direct determination of alkyl phosphates in human urine by liquid chromatography/electrospray tandem mass spectrometry

A novel and rapid procedure based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the determination of dialkyl phosphates (metabolites of organophosphorus pesticides) in human urine has been developed. After addition of 40 mM tetrabutylammonium acetate, 10 microL of urine sample were directly injected into the LC/MS/MS system. The method was validated yielding calibration curves with correlation coefficients greater than 0.997 and repeatability coefficient of variation (CV) lower than 9%. The accuracy and precision were evaluated by direct injection of spiked samples at 10 and 100 microg/L obtaining recoveries between 78 and 119% with coefficients of variation below 12%. Limits of detection of 1 microg/L for diethyl phosphate (DEP), diethylthiophosphate (DETP) and diethyldithiophosphate (DEDTP) and 2 microg/L for dimethyldithiophosphate (DMDTP) were achieved, all the analytes being detected in negative ion mode. The fragmentation pathway of dialkyl phosphates allowed us the use of an additional transition for confirmation in order to improve their identification in real-world samples. The applicability of the LC/MS/MS method was demonstrated by applying it to the analysis of urine samples of farmers exposed to the organophosphorus pesticide chlorpyrifos. Good correlation between application of the product in the field (citrus orchards), concentration levels of dialkyl phosphates and levels of the chlorpyrifos-specific metabolite (1,3,5-trichloro-2-pyridinol) was obtained.     

Quantitation of glutathione by liquid chromatography/positive electrospray ionization tandem mass spectrometry

Glutathione (GSH) is a tripeptide composed of glutamate, cysteine, and glycine. It is present in practically all cells and has several important roles, such as preventing the oxidation of the sulfhydryl groups of proteins within a cell. Evidence for GSH deficiency or depletion has been found in a variety of diseases and toxicity-related studies, including diabetes and induction of oxidative stress to form reactive oxygen species which cause DNA, lipid, and protein oxidations. A simple, selective, and sensitive analytical method for measuring low levels of GSH in biological fluids would therefore be desirable to conduct GSH deficiency or depletion-related mechanistic toxicity studies. Here a method for both low- and high-level quantitation of GSH from cultured cells and rat liver tissues via liquid chromatography/positive electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) has been developed. The lower limit of quantitation (LOQ) of the method was 5 ng/mL. The method is linear over a wide dynamic concentration range of 5.0 to 5000.0 ng/mL, with a correlation coefficient R2 > 0.99. The intra-day assay precision relative standard deviation (RSD) values for all quality control (QC) samples were < or =16.31%, with accuracy values ranging from 94.13 to 97.80%. The inter-day assay precision RSD values for all QC samples were < or =15.94%, with accuracy values ranging from 94.51 to 100.29%. With this method, low levels of GSH from diethyl maleate (DEM)-treated mouse lymphoma cells, and GSH in rat liver tissues, were quantified.     

Characterization of boldione and its metabolites in human urine by liquid chromatography/electrospray ionization mass spectrometry and gas chromatography/mass spectrometry

Boldione (1,4-androstadiene-3,17-dione) is a direct precursor (prohormone) to the anabolic steroid boldenone (1,4-androstadiene-17beta-ol-3-one). It is advertised as a highly anabolic/androgenic compound promoting muscularity, enhancing strength and overall physical performance, and is available on the Internet and in health stores. This work was undertaken to determine and characterize boldione and its metabolites in human urine, using both liquid chromatography with electrospray ionization mass spectrometry and gas chromatography with mass spectrometry and derivatization. Boldione and its three metabolites were detected in dosed human urine after dosing a healthy volunteer with 100 mg boldione. The excretion studies showed that boldione and its metabolites were detectable in urine for 48 h after oral administration, with maximum excretion rates after 1.8 and 3.6 h (boldenone case). The amounts of boldione and boldenone excreted in urine from this 100 mg dose were 34.45 and 15.95 mg, respectively.     

Simultaneous determination of low levels of methotrexate and cyclophosphamide in human urine by micro liquid chromatography/electrospray ionization tandem mass spectrometry

The aim of this study was to develop and validate a novel solid-phase extraction (SPE) liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the simultaneous determination of two antineoplastic drugs, cyclophosphamide (CP) and methotrexate (MTX), in human urine using trophosphamide as internal standard. The method showed good precision and accuracy (mean RSD 2.8% and 0.9%; bias 2.7% and 2.4% for MTX and CP, respectively). The lower limits of detection obtained, 0.2 microg/L(urine) for MTX and 0.04 microg/L(urine) for CP, were lower than the best previously reported values. The use of a 96-well SPE plate for matrix purification ensures a high throughput (50 samples/day), allowing the routine biological monitoring of CP and MTX as measures of occupational exposure at very low levels.     

Simultaneous determination of docetaxel and ketoconazole in rat plasma by liquid chromatography/electrospray ionization tandem mass spectrometry

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for simultaneous quantification of docetaxel and ketoconazole in rat plasma with paclitaxel as internal standard (IS). The analytes were extracted from rat plasma by using a liquid-liquid extraction technique with ethyl acetate and the LC separation was performed on a Cosmosil-C(18) analytical column (150 mm x 2.0 mm i.d., Nacalai Tesque Inc., Japan). The extracted samples were analyzed with LC/MS/MS, operating in selected reaction monitoring (SRM) mode. The SRM transitions of precursor ions to product ions were 830.3-->549.1 (m/z) for docetaxel, 531.2-->489.3 (m/z) for ketoconazole, and 876.7-->307.9 (m/z) for the IS. The calibration curves were linear over the range of 2-500 ng/mL for docetaxel and 50-20 000 ng/mL for ketoconazole, with coefficients of correlation above 0.999. The limits of quantification for docetaxel and ketoconazole were both 2 ng/mL. The limit of detection for each analyte was 1 ng/mL. The intra- and inter-day precision (CV) of analysis were within 7%, and the accuracy ranged from 95 to 110%. The overall recoveries for docetaxel and ketoconazole were about 89.0% and 91.1%, respectively. The total analysis time was only 9.0 min. This quantitation method was successfully applied to the simultaneous determination of docetaxel and ketoconazole in rat plasma and some potential interaction was found in the current coadministration pharmacokinetic study. This established method was also utilized in the in vitro and in vivo drug-drug interaction study of docetaxel and ketoconazole (to be published).     

Quantitative determination of rosuvastatin in human plasma by liquid chromatography with electrospray ionization tandem mass spectrometry

A simple and sensitive liquid chromatography/tandem mass spectrometry method was developed and validated for determining rosuvastatin in human plasma, a new synthetic hydroxymethylglutaryl-coenzyme A reductase inhibitor. The analyte and internal standard (IS; cilostazol) were extracted by simple one-step liquid/liquid extraction with ether. The organic layer was separated and evaporated under a gentle stream of nitrogen at 40 degrees C. The chromatographic separation was performed on an Atlantis C18 column (2.1 mm x 150 mm, 5.0 microm) with a mobile phase consisting of 0.2% formic acid/methanol (30:70, v/v) at a flow rate of 0.20 mL/min. The analyses were carried out by multiple reaction monitoring (MRM) using the precursor-to-product combinations of m/z 482 --> 258 and m/z 370 --> 288. The areas of peaks from the analyte and the IS were used for quantification of rosuvastatin. The method was validated according to the FDA guidelines on bioanalytical method validation. Validation results indicated that the lower limit of quantification (LLOQ) was 0.2 ng/mL and the assay exhibited a linear range of 0.2-50.0 ng/mL and gave a correlation coefficient (r) of 0.9991 or better. Quality control samples (0.4, 8, 25 and 40 ng/mL) in six replicates from three different runs of analysis demonstrated an intra-assay precision (RSD) 7.97-15.94%, an inter-assay precision 3.19-15.27%, and an overall accuracy (relative error) of < 3.7%. The method can be applied to pharmacokinetic or bioequivalence studies of rosuvastatin.     

Stable isotope dilution analysis of N-acetylaspartic acid in urine by liquid chromatography electrospray ionization tandem mass spectrometry

N-acetylaspartic acid (NAA) is a specific urinary marker for Canavan disease, an autosomal recessive leukodystrophy. We developed a 'dilute and shoot' stable isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS) method for determination of NAA in urine. Deuterated internal standard d(3)-NAA was added to untreated urine and the mixture was injected into the LC-MS/MS system operated in the negative ion mode. Chromatography was carried out on a C(8) minibore column using 50% acetonitrile solution containing 0.05% formic acid at a flow rate of 0.25 mL/min. The retention time was 1.6 min and the turnaround time was 2.2 min. NAA and d(3)-NAA were analyzed in multiple reaction monitoring mode. Calibrators and quality control samples were prepared in pooled control urine. The assay was linear up to 2000 micromol/L with limit of quantification at 1 micromol/L (S/N = 12). Interassay and intraassay coefficients of variation were less than 7% and recovery at three different concentrations was 98.9-102.5%. The LC-MS/MS method for NAA as described involves no extraction and no derivatization, showed no interference and gave excellent recovery with low variability and short analytical time. The method was successfully applied for the retrospective analysis of urine from 21 Canavan disease cases.     

Rapid identification of saponins in plant extracts by electrospray ionization multi-stage tandem mass spectrometry and liquid chromatography/tandem mass spectrometry

Electrospray ionization multi-stage tandem mass spectrometry (ESI-MS(n)) and liquid chromatography coupled with on-line mass spectrometry (LC/MS/MS) were applied to characterize saponins in crude extracts from Panax ginseng. The MS(n) data of the [M - H](-) ions of saponins can provide structural information on the sugar sequences of the saccharide chains and on the sapogins of saponins. By ESI-MS(n), non-isomeric saponins and isomeric saponins with different aglycones can be determined rapidly in plant extracts. LC/MS/MS is a good complementary analytical tool for determination of isomeric saponins. These approaches constitute powerful analytical tools for rapid screening and structural assignment of saponins in plant extracts.     

Simultaneous determination of water-soluble vitamins in selected food matrices by liquid chromatography/electrospray ionization tandem mass spectrometry

A rapid, simple and sensitive method based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) with an electrospray ionization (ESI) source for the simultaneous analysis of fourteen water-soluble vitamins (B1, B2, two B3 vitamers, B5, five B6 vitamers, B8, B9, B12 and C) in various food matrices, i.e. maize flour, green and golden kiwi and tomato pulp, is presented here. Analytes were separated by ion-suppression reversed-phase liquid chromatography in less than 10 min and detected in positive ion mode. Sensitivity and specificity of this method allowed two important results to be achieved: (i) limits of detection of the analytes at ng g(-1) levels (except for vitamin C); (ii) development of a rapid sample treatment that minimizes analyte exposition to light, air and heat, eliminating any step of extract concentration. Analyte recovery depended on the type of matrix. In particular, recovery of the analytes in maize flour was > or =70%, with the exception of vitamin C, pyridoxal-5'-phosphate and vitamin B9 (ca 40%); with tomato pulp, recovery was > or =64%, except for vitamin C (41%); with kiwi, recovery was > or =73%, except for nicotinamide (ca. 30%).     

Quantitative determination of E5880 in rat plasma by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

A simple and sensitive method is described for the determination of E5880 in rat plasma. The method is based on high-performance liquid chromatography/electrospray ionization mass spectrometry, using deuterated E5880 as an internal standard. Selected reaction monitoring is employed for selectivity and sensitivity, this in turn enables quantification in a short period of time (within 7 minutes) over the extended range of 0.1-1000 ng/ml with acceptable precision and accuracy. The method demonstrated to be suitable for the quantitative analysis of E5880 in rat plasma. The pharmacokinetic profile of E5880 after a single intravenous administration of E5880 was elucidated.     

Quantitative determination of ochratoxin A by liquid chromatography/electrospray tandem mass spectrometry

Mass spectrometry of ochratoxin A (OTA) and B (OTB) under electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) was studied. ESI offers higher sensitivities and less fragmentation than APCI. A sensitive LC/MS/MS method for the determination of ochratoxin A (OTA) in human plasma samples was developed. The absolute minimum detection limit was around 10-20 pg per injection, corresponding to 0.5 ppb in an injection equivalent to 20-40microg of human plasma. Ochratoxin B (OTB) was used as an internal standard and its absence in real-life samples was carefully checked before samples were spiked with the internal standard. It was found that these two ochratoxins are susceptible to sodium adduct formation. Fragment ions from the [M + H](+) and [M + Na](+) ions of both OTA and OTB were monitored in the multiple reaction monitoring mode. Three quantitative approaches, standard addition method, internal standard method (using ochratoxin B as an internal standard) and external standard method, were compared in the analysis of human blood plasma. Results from the mass spectrometric method were comparable to those from a conventional LC/fluorescence method. The LC/MS/MS method was also applied to the analysis of contaminated coffee samples.     

Improved characterization of tomato polyphenols using liquid chromatography/electrospray ionization linear ion trap quadrupole Orbitrap mass spectrometry and liquid chromatography/electrospray ionization tandem mass spectrometry

Tomato (Lycopersicon esculentum Mill.) is the second most important fruit crop worldwide. Tomatoes are a key component in the Mediterranean diet, which is strongly associated with a reduced risk of chronic degenerative diseases. In this work, we use a combination of mass spectrometry (MS) techniques with negative ion detection, liquid chromatography/electrospray ionization linear ion trap quadrupole‐Orbitrap‐mass spectrometry (LC/ESI‐LTQ‐Orbitrap‐MS) and liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) on a triple quadrupole, for the identification of the constituents of tomato samples. First, we tested for the presence of polyphenolic compounds through generic MS/MS experiments such as neutral loss and precursor ion scans on the triple quadrupole system. Confirmation of the compounds previously identified was accomplished by injection into the high‐resolution system (LTQ‐Orbitrap) using accurate mass measurements in MS, MS² and MS³ modes. In this way, 38 compounds were identified in tomato samples with very good mass accuracy (<2 mDa), three of them, as far as we know, not previously reported in tomato samples. Copyright © 2010 John Wiley & Sons, Ltd.     

Characterization of explosives by liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry using electrospray ionization and parent-ion scanning techniques

Analytical techniques for the detection of small amounts of explosives (in the picogram range) are now involved in various application. Some of them concern soil, water and air monitoring in order to face environmental problems related to improper handling procedures either in stocking or in wasting of the explosive products. Other areas are strictly related to forensic analysis of samples coming either from explosion areas where the matrix is various (metal, glass, wood, scraps), or from explosives transportation related to international terrorism. Generally speaking, for these applications the bulk of the matrix seriously interferes in the detection of the explosive analyte, which is usually present at trace levels. Unfortunately, despite some improvements, analytical techniques developed up today in this domain are still faced to two main constraints: the introduction of new products with unanticipated chemico-physical properties and the requirement of a routine and fast analytical method which can handle any matrix with a minimal clean-up and performing a sensitivity compatible either with the ever-decreasing demanded detection limit and with the ever-decreasing available specimen amount. These requirements can be fulfilled now by the new LC-MS and LC-MSMS techniques: mass spectrometry (MS) is likely an universal detector but even specific, especially when implemented in tandem MS (MSMS); LC is by far the most suitable technique to handle such a kind of compounds. Moreover, of a particular concern are some explosives which are reported to be thermally stable but difficult to dissolve. Some of the experiments on characterization of explosives [Octagen (HMX), Ethyleneglycol dinitrate (EGDN), Exogen (RDX), Propanetriol trinitrate (NG), Trinitrotoluene (TNT), N-Methyl-N-tetranitrobenzenamine (TETRYL), Dintrotoluene (DNT), Bis-(nitrooxy-methyl) propanediol dinitrate (PETN), Hexanitrostilbene (HNS), Triazido-trinitrobenzene (TNTAB), Tetranitro-acridone (TENAC), Hexa-nitrodiphenylamine (HEXYL), Nitroguanidine (NQ)] by LC-MS and LC-MSMS with the API-IonSpray source and using the Parent-Scan technique are presented.     

Determination of flomoxef in human plasma by liquid chromatography/electrospray ionization tandem mass spectrometry

A specific, sensitive, rapid and reproducible method for the determination of flomoxef in human plasma using high‐performance liquid chromatography–tandem mass spectrometry was developed and validated. Flomoxef was detected using an electrospay ionization method operated in negative‐ion mode. Chromatographic separation was performed in gradient elution mode on a Luna® C₁₈(2) column (3 μm , 20 × 4.0 mm) at a flow rate of 1 mL/min and runtime 3.5 min. The mobile phase consisted of acetonitrile and water containing 0.1% formic acid as additive. Extraction of flomoxef from plasma and precipitation of plasma proteins was performed with acetonitrile with an absolute recovery of 86.4 ± 1.6%. The calibration curve was linear with a correlation coefficient of 0.999 over the concentration range 10–5000 ng/mL and the lower limit of quantification was 10 ng/mL. The intra‐ and inter‐day precisions were <11.8%, while the accuracy ranged from 99.6 to 109.0%. A stability study of flomoxef revealed that it could be successfully analyzed at 4ºС over 24 h, but it was unstable in solutions at room temperature during short‐term storage (4 h) and several freeze–thaw cycles. Copyright © 2013 John Wiley & Sons, Ltd.     

Studies of iridoid glycosides using liquid chromatography/electrospray ionization tandem mass spectrometry

Fragmentation pathways of five iridoid glycosides have been studied by using electrospray ionization multi-stage tandem mass spectrometry (ESI-MS(n)). The first-stage MS data of the five iridoid glycosides were compared. The MS spectra showed that the adduct ions of iridoid glycosides and the formate anion were diagnostic ions to distinguish iridoid glycosides with a carboxyl group at the C-4 position or an ester group at the C-4 position. The MS fragmentation pathways of the five iridoid glycosides were also studied. Analyzing the product ion spectra of iridoid glycosides, some neutral losses were observed, such as H(2)O, CO(2) and glucose residues, which were very useful for the identification of the functional groups in the structures of iridoid glycosides. Furthermore, specific loss of one molecule of methyl 3-oxopropanoate or 3-oxopropanic acid was firstly discussed, which corresponded to the isomerization of the hemiacetal group in the structure of iridoid aglycone. According to the fragmentation mechanisms and HPLC/MS(n) data, the structures of five iridoid glycosides in a crude extract of Gardenia jasminoisdes fruit have been identified. Three compounds were compared with standards and the other two were identified as shanzhiside and genipin gentibioside by their MS(n) data without standard compounds. In order to further validate the veracity of the deduction, genipin gentiobioside was isolated from the extract of Gardenia jasminoisdes fruit using Purification Factory and was further identified by C- and H-NMR.     

Profiling histidine-containing dipeptides in rat tissues by liquid chromatography/electrospray ionization tandem mass spectrometry

The histidine-containing dipeptides carnosine (CAR) and structurally related anserine (ANS) and homocarnosine (HCAR), widely distributed in vertebrate organisms, have recently been proposed as endogenous quenchers for highly cytotoxic alpha,beta-unsaturated aldehydes generated by peroxidation. A sensitive, selective, specific and rapid liquid chromatographic/electrospray ionization tandem mass spectrometric assay was developed and validated for the simultaneous determination of these peptides in biological matrices in order to establish their plasma/tissue distribution. Samples (plasma or tissue homogenates from male rats) were prepared by protein precipitation with HClO(4) (1 : 1, v/v) containing H-Tyr-His-OH as internal standard. The supernatant was separated on a Phenomenex Sinergy polar-RP column with a mobile phase of water-acetonitrile-heptafluorobutyric acid (9 : 1 : 0.01, v/v/v) at a flow-rate of 0.2 ml min(-1), with a run time of 10 min. Detection was effected on an ion trap mass spectrometer equipped with an electrospray ionization interface operating in positive ionization mode. The acquisitions were in the multiple reaction monitoring mode using the following precursor --> product ion combinations: H-Tyr-His-OH (internal standard) m/z 319 --> 301; CAR m/z 227 --> 210 + 209; ANS m/z 241 --> 224 + 197 + 170; HCAR m/z 241 --> 156. The method was validated over the concentration range 15-1000 nmol g(-1) and the limit of quantification (LOQ) and limit of detection (LOD) were 12.5 and 4.2 pmol injected, respectively. The intra- and inter-day precisions were <10% (< or =17.47% at the LOQ) and the intra- and inter-assay accuracies were within +/-10% for all concentrations. The mapping profile in rat tissue gave the following results: the highest concentrations of CAR and ANS were found in skeletal muscles (soleus, gastrocnemius, tibialis), followed by the heart, cerebellum and brain (ANS below the LOQ). HCAR was found only in the brain and cerebellum. No histidine-containing dipeptides were detectable in plasma, liver, kidney and lung.