高效液相色谱-串联质谱法测定食品中硝磺草酮

1 引 言 硝磺草酮(Mesotrione),化学名2-(4-甲磺酰基2硝基苯甲酰基)环己烷1,3二酮,又名甲基磺草酮、硝磺酮,是瑞士先正达公司发明的玉米田芽前和苗后广谱选择性除草剂,因具有低毒性、高活性、对环境友好等特点[1,2],是近几年广泛使用的除草剂.但最近研究发现,硝磺草酮具有高效的起始活性和残留活性,长期食用含有硝磺草酮残留的食物会对人畜产生致癌作用,或引起胎儿畸形[3,4].欧盟和世界贸易组织对浆果、亚麻籽、越橘、栗草料等物质中硝磺草酮的限量为0.05 mg/kg.而美国、加拿大等国对芦笋、草杆、草料等物质中硝磺草酮的限量为0.01 mg/kg[10-13].     

Quantification of the new triketone herbicides, sulcotrione and mesotrione, and other important herbicides and metabolites, at the ng/l level in surface waters using liquid chromatography-tandem mass spectrometry

The LC/ESI/MSMS method allows the trace quantification (ng/l) of the new triketone herbicides, i.e. sulcotrione and mesotrione, and important herbicides and metabolites, in natural waters. Solid phase extraction (SPE) for sample enrichment is performed with OASIS (recoveries 94-112% for parent herbicides). Neutral and acidic compounds were analyzed separately with ESI in positive and negative mode, respectively. Quantification limits varied between 0.5 and 10 ng/l. The acidic herbicides detection was improved by a neutralizing post-column addition solution. The influence of ion suppression on quantification is discussed in detail. It is shown that we could overcome this problem and achieve reliable quantification using isotope labeled internal standards (ILIS) for every single analyte. The methods performance is illustrated with samples from a lake depth profile.     

Characterization and quantification of 10‐hydroxycamptothecine in Camptotheca acuminate and its medicinal preparation by liquid chromatography–ion trap mass spectrometry

A rapid and sensitive method for the identification and quantification of 10‐hydroxycamptothecine (HCPT) in Camptotheca acuminata Decne is described. The HCPT standard solution was directly infused into the ion trap mass spectrometers (IT/MS) for collecting the MSⁿ spectra. The electrospray ionization (ESI) mass spectral fragmentation pathway of HCPT was proposed and the ESI‐MSⁿ fragmentation behavior of HCPT was deduced in detail. The major fragment ions of HCPT were confirmed by MSⁿ in both negative ion and positive ion mode. The possible main cleavage pathway of fragment ions was studied. Quantification of HCPT was assigned in negative‐ion mode at a product ion at m/z 363 → 319 by LC‐MS. The LC‐MS method was validated for linearity, sensitivity, accuracy and precision, and then used to determine the content of the HCPT. Lastly, the LC‐MS method was successfully applied to determine HCPT in real samples of Camptotheca acuminate Decne and its medicinal preparation in the first time. Copyright © 2013 John Wiley & Sons, Ltd.     

Analysis of ceramides in cosmetics by reversed-phase liquid chromatography/electrospray ionization mass spectrometry with collision-induced dissociation

A rapid, sensitive and selective method involving reversed-phase liquid chromatography (LC) with electrospray ionization (ESI) mass spectrometry (MS) was employed for determination of commercial ceramides in cosmetics for quality control of the product formulation. Using this LC/ESI-MS technique, simultaneous separation and characterization of ceramides and an impurity substance were possible. Informative fragmentation patterns were obtained by employing LC/ESI-MS in both positive and negative ionization modes to identify the structures of both sphingoid base and N-acyl chains of ceramides, and also of an impurity. The combination of positive and negative mass spectra can be used for unambiguous confirmation of ceramides and for characterization of unknown species. In-source collision-induced fragmentation resulted in characteristic product anions for the ceramides containing a phytosphingosine moiety at m/z 267, 255 and 225, and for those with a sphingosine moiety at m/z 263 and 237, regardless of the length of the fatty acyl chains. The detection limit was about 0.5 pmol in selected-ion monitoring mode. Quantification using internal standards showed good linearity and a relative standard deviation of 4%. These ceramides were more sensitively detected in positive than in negative ion mode.     

Analysis of urinary nucleosides. I. Optimisation of high performance liquid chromatography/electrospray mass spectrometry

In order to optimise the analysis of urinary nucleosides by high performance liquid chromatography/mass spectrometry (HPLC/MS), the HPLC separation of these compounds was performed at different 'flow rates' and 0.2mL/min was found to give both a better separation and ionisation. The ionisation conditions were optimised to give the best intensity of the molecules quasi-molecular ions. The ion distribution profile and ionisation in both positive and negative mode were examined and the detection of the protonated molecule in positive mode chosen for further analysis. The limits of detection of the method developed are reported and representative LC/MS and LC/MS/MS spectra shown. Typical urinary nucleoside chromatograms are presented.     

Multi‐mycotoxin analysis in complex biological matrices using LC‐ESI/MS: Experimental study using triple stage quadrupole and LTQ‐Orbitrap

In the present study, we report the application of LC‐MS based on two different LC‐MS systems to mycotoxin analysis. The mycotoxins were extracted with an ACN/water/acetic acid mixture and directly injected into a LC‐MS/MS system without any dilution procedure. First, a sensitive and reliable HPLC‐ESI‐MS/MS method using selected reaction monitoring on a triple quadrupole mass spectrometer (TSQ Quantum Ultra AM) has been developed for determining 32 mycotoxins in crude extracts of wheat and maize. This method was operated both in positive and in negative ionization modes in two separate chromatographic runs. The method was validated by studies of spiked recoveries, linearity, matrix effect, intra‐assay precision and sensitivity. Further, we have developed and evaluated a method based on accurate mass measurements of extracted target ions in full scan mode using micro‐LC‐LTQ‐Orbitrap as a tool for fast quantitative analysis. Both instruments exhibited very high sensitivity and repeatability in positive ionization mode. Coupling of micro‐LC to Orbitrap technology was not applicable to the negatively ionizable compounds. The LC triple quadrupole MS method has proved to be stable in quantitation, as it is with respect to the matrix effects of grain samples.     

Combination of liquid chromatography/tandem mass spectrometry and gas chromatography/mass spectrometry for the detection of 21 anabolic steroid residues in bovine urine

For the detection of anabolic steroid residues in bovine urine, a highly sensitive liquid chromatographic/electrospray ionization tandem mass spectrometric (LC/ESI-MS/MS) method was developed using both positive and negative ionization. For four compounds the ESI mode was not sensitive enough and gas chromatographic/mass spectrometric GC/MS detection was therefore still necessary as a complementary method. The sample clean-up consisted of solid-phase extraction (SPE) on a C(18) column followed by enzymatic hydrolysis and a second solid-phase extraction on a combination of a C(18) and a NH(2) column. After this last SPE clean-up, the eluate was split into two equal aliquots. One aliquot was further purified and after derivatization used for GC/MS analysis. The other aliquot was analyzed with LC/MS/MS in both ESI+ and ESI- modes. The method was validated according to the European Commission Decision 2002/657/EC. Decision limits (CCalpha) were between 0.16 and 1 ng ml(-1) for the compounds detected with the LC/MS/MS method. The developed method is used in routine analysis in our laboratory.     

Determination of pharmaceuticals in aqueous samples using positive and negative voltage switching microbore liquid chromatography/electrospray ionization tandem mass spectrometry

Analytical methods were developed for atorvastatin, novobiocin and roxithromycin using microbore liquid chromatography/electrospray ionization tandem mass spectrometry (microbore LC/ESI-MS/MS) in positive and negative voltage switching mode. Atorvastatin and roxithromycin require the positive-ion mode, whereas the negative-ion mode is required for the determination of novobiocin. Using the positive and negative voltage switching function, the three analytes were determined with one injection, and the time required was half that required using separately run positive- and negative-ion modes, without any reduction in sensitivity. A microbore LC column (100 x 1.0 mm i.d.) was chosen for chromatographic separation with mobile phase solvents acetonitrile and 10 mM aqueous ammonium acetate. The flow-rate was 0.1 ml min(-1) and the injection volume was 1 micro l. The analytes were quantified in the multiple reaction monitoring mode with external standards. By switching the positive and negative voltage, the three analytes were determined with a 4 min chromatographic run and with instrumental detection limits of 1-3 pg. This analytical method, using a microbore LC column combined with solid-phase extraction, was applied successfully to the determination of trace levels of the above pharmaceuticals in aqueous samples. Atorvastatin was detected in a sewage treatment plant final effluent.     

Determination of estrogens and progestogens by mass spectrometric techniques (GC/MS, LC/MS and LC/MS/MS)

Steroid sex hormones and related synthetic compounds have been shown to provoke alarming estrogenic effects in aquatic organisms, such as feminization, at very low concentrations (ng/L or pg/L). In this work, different chromatographic techniques, namely, gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS), are discussed for the analysis of estrogens, both free and conjugated, and progestogens, and the sensitivities achieved with the various techniques are inter-compared. GC/MS analyses are usually carried out after derivatization of the analytes with bis(trimethylsilyl)trifluoroacetamide (BSTFA). For LC/MS and LC/MS/MS analyses, different instruments, ionization techniques (electrospray (ESI) and atmospheric pressure chemical ionization (APCI)), ionization modes (negative ion (NI) and positive ion (PI)) and monitoring modes (selected ion monitoring (SIM) and selected reaction monitoring (SRM)) are generally employed. Based on sensitivity and selectivity, LC/ESI-MS/MS is generally the method of choice for determination of estrogens in the NI mode and of progestogens in the PI mode (instrumental detection limits (IDLs) 0.1-10 ng/mL). IDLs achieved by LC/ESI-MS in the SIM mode and by LC/ESI-MS/MS in the SRM mode were, in general, comparable, although the selectivity of the latter is significantly higher and essential to avoid false positive determinations in the analysis of real samples. Conclusions and future perspectives are outlined.     

Simultaneous detection of type A and type B trichothecenes in cereals by liquid chromatography-electrospray ionization mass spectrometry using NaCl as cationization agent

A LC/MS method for the simultaneous determination of both type A and type B trichothecenes by using an electrospray ionization (ESI) interface in the positive ionization mode with a single quadrupole analyzer is described. In order to enhance the ionization of both groups of trichothecenes, the sodium ion was used as cationization agent by adding sodium chloride to the eluent. All LC/MS parameters were optimized. The newly developed LC/ESI-MS method was applied to the analysis of a wheat reference material and cereal-based foods and feeds.     

Ion trap liquid chromatography/tandem mass spectrometry analysis of leukotriene B4 in exhaled breath condensate

The objective of this study is the measurement of leukotriene B7 (LTB4), a potent inflammatory mediator, in exhaled breath condensate by using liquid chromatography/mass spectrometry (LC/MS and LC/MS/MS). Condensation of exhaled breath is a non-invasive method to collect airway secretions. Deuterated (d4)-LTB4 was used as internal standard. The MS and MS/MS behavior of LTB4 and LTB4-d4 was studied by electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) in both positive and negative ion polarity mode. Preliminary results show that monitoring negative ions in ESI mode has the best sensitivity for both LTB4 and LTB4-d4. Therefore, negative ESI was chosen, and the [M-H]- ions at m/z 335 and 339 were selected for quantification. The lower limit of quantification for LTB4, expressed as the lowest point of the calibration curve, was 100 pg/mL. Using this technique, we measured LTB4 in exhaled breath condensate in two healthy subjects, four asthmatic patients on anti-inflammatory treatment, and four asthmatic patients who were not on anti-inflammatory drugs. Exhaled LTB4 concentrations were detected only in asthmatic patients who were not on anti-inflammatory therapy. This method is potentially useful for non-invasive assessment of airway inflammation, but the sensitivity of the technique needs to be improved.     

ESI-MS/MS analysis of underivatised amino acids: a new tool for the diagnosis of inherited disorders of amino acid metabolism. Fragmentation study of 79 molecules of biological interest in positive and negative ionisation mode

The diagnosis of inherited disorders of amino acids (AA) metabolism is usually performed on automated analysers by ion-exchange chromatography and quantification after ninhydrin derivatisation of about 50 different AA. A single run liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for these molecules can be an alternative to this time-consuming technique. The first step of this development is the infusion study of the fragmentation of 79 molecules of biological interest in electrospray ionisation tandem mass spectrometry (ESI-MS/MS), in positive and in negative ionisation mode. Among them, three molecules can be detected only in negative ionisation mode, 38 only in positive mode and 38 in the two modes. All the most abundant fragmentations are presented, with optimisation of the MS/MS parameters. The positive ionisation mode was retained for the simultaneous analysis of 76 molecules. One sensitive and/or specific transition is proposed for the monitoring of each molecule. Improvement in sensitivity of detection was obtained with the use of an acidic mobile phase. Flow injection analysis studies led us to highlight a number of interferences-due to isobaric molecules, to in-source collision-induced dissociation, or to natural isotopic distribution of the elements-which are listed. For a reliable quantification method, these molecules have to be separated by LC before analysis in the tandem mass spectrometer. Ion-pairing reversed-phase liquid chromatography (RPLC) using perfluorinated carboxylic acids as ion-pairing agents has already been found suitable for analysis of AA in MS/MS positive ionisation mode and is under development.     

Optimization of a liquid chromatography method based on simultaneous electrospray ionization mass spectrometric and ultraviolet photodiode array detection for analysis of flavonoid glycosides

Different reversed-phase liquid chromatography (LC) columns of conventional dimensions were coupled to an ultraviolet photodiode array detector (UV-DAD) and a magnetic sector-type spectrometer, equipped with an electrospray ionization (ESI) source, by a laboratory-made flow splitter. A mixture of three flavonoid-O-glycosides was employed to examine the effects of the solvent composition, the flow rate, the stationary phase, the pH and the organic acid added, on the chromatographic separation, the UV-DAD detection, the ESI process and the entire LC system with ESI-MS and UV-DAD detection. In the positive ion mode, methanol containing 1% acetic acid was by far the most sensitive in ESI-MS analysis, whereas an acetonitrile/water mobile phase containing 0.5% formic acid was proved to give the best sensitivity in LC/ESI-MS/UV-DAD analysis. In the negative ion mode, the highest sensitivity was obtained with a mobile phase containing 0.1% formic acid, while addition of bases decreased the sensititvity. The optimal flow rate was higher in negative ESI (20-50 micro L/min) than in positive ESI (5 micro L/min), and the percentage of organic phase had an influence on the sensitivity of ESI-MS detection. With regard to the selection of a suitable C(18) reversed-phase LC column, a column which is well end-capped is to be preferred, because residual silanol groups appear to impair the separation of flavonoid glycosides. The optimized LC/ESI-MS/UV-DAD method was applied to a commercial Crataegus extract, which is used in phytomedicine to treat cardiovascular problems and is known to be rich in flavonoids. It is demonstrated how UV spectra and first-order ESI mass spectra allow a fast characterization of flavonoids, even if reference compounds are not available or at hand.     

Liquid chromatography/tandem mass spectrometry for the identification and determination of trichothecenes in maize

A reliable, sensitive and selective liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed to determine four trichothecene mycotoxins (nivalenol, deoxynivalenol, fusarenon X and 3-acetyldeoxynivalenol) in maize. Sample preparation was performed by extracting the analytes with a mixture of acetonitrile and water, followed by a solid-phase extraction with Carbograph-4 cartridges as the purification step. For the LC/MS/MS analysis two interfacing systems, Turbo IonSpray (TISP) and atmospheric pressure chemical ionization (APCI), were compared in both negative and positive ion modes. LC and MS parameters were optimized to achieve better results and sensitivity. The effect of mobile phase modifiers such as ammonium acetate and formic acid on the ionization yield was also evaluated. The best results were obtained using the electrospray ionization (ESI) interface in negative ion mode and the multiple reaction monitoring mode (MRM) for the quantitation. The detection limits ranged between 10 ng/g for fusarenon X and 1.5 ng/g for deoxynivalenol. A linear working range was achieved with a standard deviation between 3 and 10% and recovery rates from the maize samples above 81%. The procedure was applied to the analysis of a set of maize samples collected from farms located in different areas of northern and central Italy. The investigated samples turned out to be contaminated primarily with deoxynivalenol and, to a minor extent, with its derivatives.     

Comparison of atmospheric pressure chemical ionization and electrospray ionization mass spectrometry for the detection of lignans from sesame seeds

In sesame seeds, high concentrations of lignans are present. When these lignans are fermented in the human colon, a range of structurally different lignans is formed. A good liquid chromatography/mass spectrometry (LC/MS) protocol for the analysis of lignans in complex mixtures is lacking. In order to develop such a protocol, electrospray ionization (ESI)-MS and atmospheric pressure chemical ionization (APCI)-MS, both in the positive and negative ionization mode, were compared. An extract from defatted sesame meal was analyzed by APCI-MS and ESI-MS, before and after deglucosylation. APCI-MS was found to be a more generic method than ESI-MS because lignans, especially sesamolin, sesamin and pinoresinol, were better detected by APCI-MS than by ESI-MS. Positive and negative ionization modes had to be combined in order to detect all lignans in a bacterial culture grown on aglyconic, acid-treated lignans from sesame oil and defatted sesame meal. Lignans with methylenedioxy-bridged furanofuran structures mostly lack phenolic hydroxyl groups and were, therefore, optimally detected in positive ionization mode. Dibenzylbutadiene lignans, which were formed during fermentation, carry hydroxyl groups and were better detected in negative ionization mode.     

Development and validation of a liquid chromatographic/tandem mass spectrometric assay for the quantitation of nucleoside HIV reverse transcriptase inhibitors in biological matrices

Besides liquid chromatographic (LC)/UV methods adapted to therapeutic drug monitoring, there is still a need for more powerful techniques that can be used for pharmacological research and clinical purposes. We developed an LC method coupled with tandem mass spectrometry (MS/MS) to separate, detect and quantify with high sensitivity the nucleoside analogues used in multitherapies (zidovudine, stavudine, zalcitabine, didanosine, lamivudine and abacavir) in plasma and in the intracellular medium. We worked on two essential issues: (i) the need to use two ionization modes in order to achieve the best sensitivity, which leads to the optimization of the chromatographic separation of drugs detected in the positive ionization mode and drugs detected in the negative ionization mode, and (ii) the need to optimize the extraction step in order to enhance sample recovery. The peripheral blood mononuclear cells were lysed in Tris buffer-MeOH. A clean-up procedure was performed by solid-phase extraction only for plasma samples. The LC separation was carried out on a Zorbax Stable Bond C(18) column followed by MS/MS analysis after electrospray ionization in either the negative or positive mode. The positive ionization mode was applied at the beginning of the run to detect zalcitabine and lamivudine, then the ionization mode was changed to negative for the detection of didanosine, stavudine, internal standard and zidovudine. The calibration range for all the analytes was 0.5-200 ng ml(-1). The recoveries were between 64 and 90%, with coefficients of variation (CVs) lower than 15%. The inaccuracy (bias) was +/-15% with CVs always lower than 12%. The analytes were stable at room temperature and in the extraction solvent for at least 24 h, after storage at -80 degrees C for 3 months, after three freeze-thaw cycles and in the injection solvent after 48 h at 4 degrees C. Together with the measurement of intracellular triphosphorylated metabolites thanks to the powerful plasma and intracellular assay method for intact drugs, it is possible to describe the behaviour of nucleoside analogues against HIV through plasma pharmacokinetics, cell membrane diffusion including drug transport involvement, and also the intracellular metabolism.     

A rapid and sensitive liquid chromatography/negative ion tandem mass spectrometry method for the determination of an indolocarbazole in human plasma using 96-well diatomaceous earth plates for solid-liquid extraction [correction of using internal standard (IS) 96-well diatomaceous earth plates for solid-liquid extraction

A sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the quantitation of a novel topoisomerase I inhibitor (indolocarbazole derivative I) in human plasma was developed to support clinical studies. Drug and internal standard were isolated from plasma by solid-liquid extraction using 96-well diatomaceous earth plates. Various extraction solvents were evaluated for extraction of I and 9% isopropyl alcohol (IPA) in methyl-tert-butyl ether (MtBE) was chosen as the optimal extraction solvent. The sensitivity of this LC/MS/MS method is 10x higher in negative ion mode using alkaline conditions than in positive ion mode using a wide range of pH's. A mobile phase with 2 mM ammonium hydroxide enhanced the sensitivity in negative ion mode over other volatile bases. The calibration curve for compound I is linear over the range 0.05-200 ng/mL in plasma and the lower limit of quantification (LLOQ) of the assay is 0.05 ng/mL, when 0.25 mL of plasma is processed. The method was fully validated and successfully applied to plasma samples from clinical studies. Performing chromatography at high pH, for enhanced negative ion sensitivity, eliminates the need for post-column addition of base. Furthermore, the 96-well diatomaceous earth plate extraction offers the following advantages over liquid-liquid extraction (LLE) or solid-phase extraction (SPE): clean sample extracts with reduced sample preparation time; increased sample throughput; no conditioning or washing steps; and a neutral eluate applicable to acid/base labile compounds.     

Applicability of gradient liquid chromatography with tandem mass spectrometry to the simultaneous screening for about 100 pesticides in crops

A method was developed for screening crops for a range of pesticide residues by liquid chromatography/tandem mass spectrometry (LC/MS/MS). A complete set of LC, electrospray ionization (ESI), and tandem MS acquisition parameters was established for the determination of 108 analytes; these parameters were used for the simultaneous acquisition of 98 analytes in the positive ESI mode and 10 analytes in an additional MS/MS method in the negative ESI mode. The entire procedure involves extraction of residues with methanol-water and partition into dichloromethane. The utility of the method is demonstrated by the analysis of crops of 5 matrix types (water-containing, acidic, dry, sugar-containing, and fatty). Of 108 pesticides/metabolites tested, 104 showed sufficient stability in most matrixes for determination by LC/MS/MS. These analytes belong to 20 chemical classes, which demonstrate the general applicability of the method for multiclass analysis. By using matrix-matched standards, 67 compounds could be determined in most matrixes with recoveries of 70-120% and a relative standard deviation of < or = 25% at the 0.01 mg/kg level.     

Structure elucidation of four related substances in gramicidin with liquid chromatography/mass spectrometry

A selective reversed phase liquid chromatography/mass spectrometry (LC/MS(n)) method is described for the identification of related substances in commercial gramicidin samples. Mass spectral data are acquired on an LCQ ion trap mass spectrometer equipped with an electrospray interface operated in the positive and the negative ion mode. The LCQ is ideally suited for identification of related substances because it provides on-line LC/MS(n) capability. Compared with UV detection the main advantage of this hyphenated LC/MS(n) technique is the efficient identification of novel related substances without time-consuming isolation and purification procedures. Using this method four novel related substances were separated and identified in a commercial sample.     

Simultaneous determination of amiloride and hydrochlorothiazide in human plasma by liquid chromatography/tandem mass spectrometry with positive/negative ion-switching electrospray ionisation

A new method for simultaneous determination of amiloride and hydrochlorothiazide by liquid chromatography/electrospray tandem mass spectrometry (LC/MS/MS) operated in positive and negative ionization switching mode was developed and validated. Protein precipitation with acetonitrile was selected for sample preparation. The analytes were separated on a Phenomenex Curosil-PFP (250x4.6 mm, 5 microm) column by a gradient elution with a mobile phase consisting of 0.15% formic acid solution containing 0.23% ammonium acetate and methanol pumped at a flow rate of 1.0 mL.min(-1). Rizatriptan was used as the internal standard (IS) for quantification. The determination was carried out on a Waters Quattro-micro triple-quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) mode using the following transitions monitored simultaneously: positive m/z 230-->171 for amiloride, m/z 270-->158 for rizatriptan, and negative m/z 296-->205 for hydrochlorothiazide. The lower limits of quantification (LLOQs) were 0.1 and 1.0 ng.mL(-1) for amiloride and hydrochlorothiazide, respectively, which were lower than other published methods by using ultraviolet (UV), fluorimetric or mass spectrometric detection. The intra- and inter-day precision and accuracy were studied at three different concentration levels and were always better than 15% (n=5). This simple and robust LC/MS/MS method was successfully applied to the pharmacokinetic study of compound amiloride and hydrochlorothiazide tablets in healthy male Chinese volunteers.