Multiresidue Analysis of Pesticides in Vegetables and Citrus Fruits by LC–MS–MS

In this work, an analytical multiresidue method using liquid chromatography tandem-mass spectrometry (LC–MS–MS) with triple quadrupole in selected reaction monitoring (SRM) mode for the simultaneous determination of 54 pesticides in vegetables (pepper and tomato) and citrus fruits (orange and lemon) has been developed. The procedure involves initial single phase extraction of sample with acetonitrile by agitation, followed by liquid–liquid partition aided by “salting out” process using NaCl. The average recovery by the LC–MS–MS method obtained for these compounds varied from 65.5 to 114.5% with a relative standard deviation between 2.3 and 8.3%. The method presents good linearity over the range assayed 10–500 μg L^?1 (except famoxadone 50–1,000 μg L^?1) and the detection limits for the pesticides studied varied from 0.03 to 14.9 μg kg^?1. The proposed method was used to determine pesticide levels in vegetables and citrus fruit samples from different experimental orchards and greenhouses from the Region of Murcia.     

Analysis of Linarin and Its Metabolites in Rat Urine by LC–MS/MS

Liquid chromatography–ion trap mass spectrometry was employed to investigate the metabolism of linarin in rats. Identification and structural elucidation of the metabolites were performed by comparing the differences in molecular masses, retention times, and full scan MS ⁿ spectra between linarin and its metabolites. Six metabolites (acacetin, apigenin, acacetin glucuronide, apigenin glucuronide, acacetin sulfate, apigenin sulfate) were detected in rat urine after oral administration of linarin at the dose of 50 mg kg^?1. Furthermore, a selective and sensitive liquid chromatography–triple quadruple mass spectrometry assay was developed and validated for the simultaneous determination of linarin and acacetin (the major metabolite of linarin) in rat urine. Chromatographic separation was carried out on a C₁₈ column, and mass spectrometric detection was performed using a triple-quadrupole mass spectrometer coupled with an electrospray ionization source in the positive ion mode. Quantitation of linarin and acacetin was performed using selected reaction monitoring of precursor–product ion transitions at m/z 593 → 285 for linarin, 285 → 242 for acacetin, and 303 → 153 for hesperitin (internal standard), respectively. The assay exhibited good linearity (r > 0.9900) for both linarin and acacetin. The intra- and inter-day precisions were <13.4 % and the accuracy was between ?8.1 and 3.1 %. The method was successfully applied to the urinary excretion study of linarin in rats after oral administration of linarin.     

Determination of Pitavastatin in Human Plasma by LC–MS–MS

A simple and rapid LC–MS–MS assay was developed and validated for the quantitative determination of pitavastatin in human plasma. Sample pretreatment involved simple protein precipitation by addition of acetonitrile. Separation was on an Agilent 1.8 μm Zorbax SB-C18 column (150 mm × 4.6 mm) at 25 °C using isocratic elution with methanol–0.1% formic acid in water (85:15, v/v) at a flow rate of 0.4 mL min^?1. Detection was performed using electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the ion transitions m/z 422.0 → 290.1 for pitavastatin, and m/z 330.1 → 192.1 for paroxetine (IS). LC–MS–MS was found to improve the quantitation of pitavastatin in plasma and was successfully applied in pharmacokinetic studies.     

Analysis of Deoxynivalenol,Nivalenol, Zearalenone in Food by LC–APCI–MS

The detection of mycotoxins is an important task for analytical analysis, as they are a source of contaminants in foods today. The very small amounts of toxic mycotoxins (zearalenone, deoxynivalenol) make it important to determine the most reliable analytical methods. There are several options for the detection of mycotoxins, LC–API–MS techniques being the most common ones. The aim of the present determination is to give an overview on the application of LC–(API)-MS in the analysis of frequently occurring and highly toxic mycotoxins, such as deoxynivalenol, nivalenol and zearalenone, in organic foods. The limits of these three toxins in foods are very low: deoxynivalenol 1,250 μg kg^?1, nivalenol 0.9 μg kg^?1 of body weight, zearalenone 100 μg kg^?1.     

Simultaneous Quantitative Analysis of Shikonin and Deoxyshikonin in Rat Plasma by Rapid LC–ESI–MS–MS

The purpose of this article was to develop a rapid and robust LC–MS–MS method for quantifying shikonin and deoxyshikonin simultaneously in rat plasma using emodin as internal standard. The LC system consisted of an Agilent ZORBAX SB-C18 (1.8 μm, 250 × 4.6 mm, 20 °C) column. Elution with an isocratic mobile phase consisted of methanol/10 mM ammonium acetate in water/acetonitrile containing 0.05% formic acid (45:10:45, v/v/v) at a flow rate of 0.8 mL min^?1 yielded sharp, high-resolved peaks within 12 min. The lower limits of quantitation were 0.5 ng mL^?1 for shikonin, and 8 ng mL^?1 for deoxyshikonin. Correlation coefficient (r) values for the linear range of two analytes were greater than 0.99. Assay precision was <13% and accuracy was 87–99%. This newly developed method was used to the pharmacokinetic studies of the shikonin analogues in rats after intravenous administration (n = 4).     

Quantitative Analysis of Fudosteine in Human Plasma Using a Simple Precipitation Step Followed by LC–MS–MS

Fudosteine [(-)-(R)-2-amino-3-(3-hydroxypropylthio) propionic acid] is a synthetic derivative of l-cysteine as a novel expectorant. To facilitate pharmacokinetics studies of fudosteine in man, a sensitive and specific LC–MS–MS method for the quantitative detection of fudosteine in human plasma was developed and validated. The method involved the addition of fosfomycin as internal standards, protein-precipitation, HPLC separation, and quantification by MS/MS system using negative electrospray ionization in the multiple reaction monitoring mode. The precursor → product ion transitions were monitored at 177.6 → 90.9 and 136.8 → 78.9 for fudosteine and the IS, respectively. The lower limit of quantitation (LLOQ) was 5 ng mL⁻¹. The calibration curves for fudosteine was linear over a concentration range of 0.005–10 µg mL⁻¹. The intra- and inter-day analyses of QC samples at 0.01, 1, 10 µg mL⁻¹ indicated good precision %RSD between 92.00 and 101.02%. The fudosteine was stable in human plasma stored at room temperature for at least 12 h, 4 °C for at least 12 h, −20 °C for at least 20 days, and at least three freeze–thaw cycles procedures. This accurate and highly specific assay provides a useful method for evaluating the pharmacokinetics of fudosteine in human.

     

Determination of Eleutherosides in Dietary Supplements by LC–MS/MS

A simple and specific method for the simultaneous determination of eleutherosides B and E in powdered rhizomes of Eleutherococcus senticosus extract and in solid and liquid dietary supplements was developed and validated. E. senticosus extracts, often mixed with other plants or herbal extracts, are widely used in food supplements because of the tonic and adaptogenic activities referred to the eleutherosides B and E. In this study, samples were analyzed by a liquid chromatography-electrospray tandem mass spectrometry (LC–ESI-MS/MS) method operated in single reaction monitoring (SRM). Validation was carried out in terms of limit of detection (LOD), limit of quantitation (LOQ), linearity, precision and trueness. LOD and LOQ values were fixed at 3 μg L^?1 and 10 μg L^?1, respectively, whereas linearity was established within 10–1,000 μg L^?1 range for both compounds. Good precision was obtained for both eleutherosides in terms of intra-day precision (RSD % lower than 4 %) and inter-day precision (RSD % lower than 6 %). Good percentage recoveries were obtained for both eleutherosides (91.5–103.6 %). Finally, the developed method was successfully applied to analyze a number of solid and liquid commercial dietary supplements containing E. senticosus extracts, also mixed with other herbal extracts.     

Determination of Amphotericin B in Human Cerebrospinal Fluid by LC–MS–MS

A simple, specific and sensitive column liquid chromatography–tandem mass spectrometry method has been developed for the determination of amphotericin B in human cerebrospinal fluid. Samples were prepared by dilution with methanol and quantitated by MS–MS detection in the positive mode. The determination was validated in the concentration range of 0.5–100 ng mL^?1 using 100 μL of human cerebrospinal fluid. The method was successfully used to support routine therapeutic drug monitoring.     

Determination of Fenofibric Acid in Human Plasma by LC–MS/MS and LC–UV

A sensitive, high-throughput and economic liquid chromatographic method for determination of fenofibric acid in human plasma was developed and validated by ultraviolet detection and tandem mass spectrometry, then applied in pharmacokinetic study to investigate Lipanthyl™ 200 mg MC bioavailability under food and fasting conditions. Fenofibric acid with 2-chloro fenofibric acid-d6 (internal standard) was extracted from 100 µL of human plasma by acetonitrile in a single extraction step. 25 and 2 µL from supernatant were injected onto ACE column, 50 mm, 5 micron with 4.6 mm inner diameter for LC–UV and 2.1 mm for LC–MS/MS, and both systems were eluted isocratically by water:methanol:formic acid (35:65:0.1, v/v/v), with a constant flow rate of 1 mL min⁻¹. The established calibration curve was linear between 0.05–20 µg mL⁻¹, and the within- and between-day precisions were all below 13 % in both LC–MS/MS and LC–UV systems during validation, and accuracies ranged between 91 and 112 %. Twenty-eight healthy adult subjects participated in this clinical study, and the pharmacokinetic parameters including coefficient of variation were calculated and discussed. A dramatic decrease in C _max and AUC0-72 (3.63- and 1.85-fold, respectively) were observed for Lipanthyl™ MC under fasting conditions with more variable inter subject measurements comparing to the fed state.

     

Analysis of Saffron (Crocus sativus L. Stigma) Components by LC–MS–MS

A liquid chromatographic tandem mass spectrometric validated method was developed for the detection of chemicals attributing color, flavor, taste and medicinal properties to saffron (Crocus sativus L. stigma). Ultrasonic extractions of saffron stigmas were followed by LC procedure with Pinnacle II Cyano (5 μm 150 × 2.1 mm) column and acetonitrile: water (70:30, v/v) as mobile phase. Deprotonated ions formed by a turbo ion spray in negative MS mode were used to detect the analytes. MS–MS detection was by monitoring precursors (m/z) fragmentations; of 149 → 113 (safranal), 327 → 283 (crocetin), 329 → 167 (picrocrocin), 355 → 327 (dimethyl crocetin), 489 → 327 (crocin E), 535 → 489 (carotenes), 651 → 327 (crocin C), 813 → 652 (crocin B), 975 → 651 (crocin A) and 1,137 → 813 (crocin F). The method was validated for linearity, precision, repeatability and specificity.     

Analysis of neem oils by LC–MS and degradation kinetics of azadirachtin-A in a controlled environment

Since it was first isolated, the oil extracted from seeds of neem (Azadirachtin indica A juss) has been extensively studied in terms of its efficacy as an insecticide. Several industrial formulations are produced as emulsifiable solutions containing a stated titer of the active ingredient azadirachtin-A (AZ-A). The work reported here is the characterization of a formulation of this insecticide marketed under the name of Neem-azal T/S and kinetic studies of the major active ingredient of this formulation. We initially performed liquid–liquid extraction to isolate the neem oil from other ingredients in the commercial mixture. This was followed by a purification using flash chromatography and semi-preparative chromatography, leading to ¹³C NMR identification of structures such as azadirachtin-A, azadirachtin-B, and azadirachtin-H. The neem extract was also characterized by HPLC–MS using two ionization sources, APCI (atmospheric pressure chemical ionization) and ESI (electrospray ionization) in positive and negative ion modes of detection. This led to the identification of other compounds present in the extract—azadirachtin-D, azadirachtin-I, deacetylnimbin, deacetylsalannin, nimbin, and salannin. The comparative study of data gathered by use of the two ionization sources is discussed and shows that the ESI source enables the largest number of structures to be identified. In a second part, kinetic changes in the main product (AZ-A) were studied under precise conditions of pH (2, 4, 6, and 8), temperature (40 to 70 °C), and light (UV, dark room and in daylight). This enabled us to determine the degradation kinetics of the product (AZ-A) over time. The activation energy of the molecule (75±9 kJ mol^–1) was determined by examining thermal stability in the range 40 to 70 °C. The degradation products of this compound were identified by use of HPLC–MS and HPLC–MS–MS. The results enabled proposal of a chemical degradation reaction route for AZ-A under different conditions of pH and temperature. The data show that at room temperature and pH between 4 and 5 the product degrades into two preferential forms that are hydrolyzed to a single product over time and as a function of pH change.     

Analysis of Volatile Compounds in Radix Bupleuri Injection by GC–MS–MS

Static headspace sampling, headspace solid-phase microextraction, and direct immersion solid-phase microextraction coupled with gas chromatography–tandem mass spectrometry have been developed for determination of the volatile components in Radix bupleuri injection. A total of 78 compounds were identified from Radix bupleuri injection. Direct immersion solid-phase microextraction gave a better extraction efficiency for polar compounds, including organic acids and alcohols, than headspace solid-phase microextraction or static headspace sampling. Product ion isotope pattern analysis was applied to determine the elemental composition of the precursor ion, which could make the qualitative analysis more accurate and reliable.     

Simultaneous Determination of Tamsulosin and Dutasteride in Human Plasma by LC–MS–MS

A sensitive and selective liquid chromatographic tandem mass spectrometric (LC–MS–MS) method was developed for simultaneous identification and quantification of tamsulosin and dutasteride in human plasma, which was well applied to clinical study. The method was based on liquid–liquid extraction, followed by an LC procedure with a Gemini C-18, 50 mm × 2.0 mm (3 μm) column and using methanol:ammonium formate (97:3, v/v) as the mobile phase. Protonated ions formed by a turbo ionspray in positive mode were used to detect analytes and internal standard. MS–MS detection was by monitoring the fragmentation of 409.1 → 228.1 (m/z) for tamsulosin, 529.3 → 461.3 (m/z) for dutasteride and 373.2 → 305.3 (m/z) for finasteride (IS) on a triple quadrupole mass spectrometer. The lower limit of quantification for both tamsulosin and dutasteride was 1 ng mL^?1. The proposed method enables the unambiguous identification and quantification of tamsulosin and dutasteride for clinical drug monitoring.     

Simultaneous Analysis of Selected Typical Antibiotics in Manure by Microwave-Assisted Extraction and LC–MS n

Analysis of antibiotics in the environment has become an urgent issue. A novel method entailing microwave-assisted extraction (MAE) and high-performance liquid chromatography–electrospray mass spectrometry (LC–MS ⁿ ) has been developed for determination of selected typical antibiotics, including quinolones, sulfonamides, and tetracyclines, in manure. Compared with ultrasonic extraction, MAE significantly increased recovery of fluoroquinolones (63–106%), sulfonamides (64–133%), and tetracyclines (64–109%) from manure. Acetonitrile acidified with formic acid buffer solution (pH 4.0) was used for extraction of swine manure whereas chicken manure was extracted with 0.1 M EDTA–McIlvaine buffer solution. Limits of quantification (LOQ) for all compounds were in the range 5.12–168.4 μg kg^?1 dry matter, which were satisfactory for analysis of all samples. The suitability of the method was assessed by analysis of manure from six different sites.     

Simultaneous Determination of Atenolol and Chlorthalidone by LC–MS–MS in Human Plasma

A simple, sensitive, selective and rapid liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous separation and quantitation of atenolol and chlorthalidone in human plasma using metoprolol and hydrochlorothiazide as internal standard. Following solid phase extraction, the analytes were separated by an isocratic mobile phase on a reversed-phase C₁₈ column and analyzed by MS in the multiple reaction-monitoring mode (atenolol in positive and chlorthalidone in the negative ion mode). The limit of quantitation for this method was 10 and 15 ng mL^?1 and the linear dynamic range was generally 10–2,050 ng mL^?1 and 15–3,035 ng mL^?1 for atenolol and chlorthalidone, respectively.     

Pressurized-liquid extraction for determination of illicit drugs in hair by LC–MS–MS

An LC–MS–MS-based procedure for determination in hair of 14 different drugs of abuse belonging to the classes cocaine, amphetamine-like compounds, opiates, and hallucinogens has been developed. A pressurized-liquid extraction procedure was used and proved useful for quantitative recovery of all the analytes tested. This procedure, in conjunction with a simple decontamination step, performed to avoid false-positive samples, enabled the detection of all the analytes with LOQ ranging from 1.8 to 16 pg mg^?1 and accuracy varying from 85 to 111 %. The procedure was validated in accordance with the SOFT/AAFS guidelines and seems to be suitable for routine determination of the drugs tested in hair.     

Fingerprint Analysis of Euonymus alatus (Thuhb) siebold by LC–DAD and LC–ESI–MS

A liquid chromatography coupled with photodiode array detection and electrospray ionization tandem mass spectrometry method was first developed for a chemical fingerprint analysis of Euonymus alatus (Thuhb) siebold (EAS) and rapid identification of major compounds in the fingerprints. Fingerprint profiles were found to be consistent for the herbs acquired from different locations, but the relative abundance of peaks was varied. Twelve peaks were chosen as the common peaks. Quercetin and rutin were detected by comparing the retention times, MS and UV spectra with the standards. The relative retention time and relative peak area of the 12 peaks in the fingerprint were calculated by setting the quercetin as the reference peak. The experimental data were used for similarity calculation and hierarchical clustering analysis. By comparing the UV and MS spectra data with those of the authentic standards and literature, five main peaks in the fingerprints were identified. Finally, five medicinal portions of the herb (leaf, fruit, stem, pterygium and root) were also analyzed by this method. It was found that there were similar chemical components in different parts of this herb but the contents were very different. The developed fingerprint assay was specific and could be readily utilized for comprehensive evaluation of EAS, as well as to distinguish different medicinal portions.     

Analysis of Biologically Active Constituents in Centella asiatica by Microwave-Assisted Extraction Combined with LC–MS

Centella asiatica (L.) Urban is a traditional herbal medicine used in Asiatic countries, and is commonly used to treat various wounds, leprosy, tuberculosis and lupus diseases. In this work, a new method based on microwave assisted extraction followed by liquid chromatography–diode array detection–electrospray ionization multistage tandem mass spectrometry analysis has been developed for the identification and quantification of biologically active constituents in C. asiatica, including asiatic acid, asiaticoside and madecassoside. The separation was performed on an Agilent Eclipse C₁₈ column (150 mm × 4.6 mm i.d., 5 μm) with gradient elution of acetonitrile and 0.1% aqueous acetic acid within 50 min. Detection was performed at 205 nm. The calibration curves showed good linearity (r ² > 0.9992). The limits of detection ranged from 1.2 to 1.6 μg mL^?1. The intra- and inter-day precision was less than 3% and the recovery of the assay was in the range of 95.4–106.8%. The method was successfully applied to the quantification of the three constituents in different samples of C. asiatica. The results indicated that the developed method could be considered to be a simple, rapid and reliable method for the quality evaluation of C. asiatica. The samples were also analyzed on a liquid chromatography–electrospray ionization–time-of-flight mass spectrometry system to confirm the identification results.     

LC–MS–MS Analysis of Mirtazapine in Plasma, and Determination of Pharmacokinetic Data for Rats

A sensitive LC–MS–MS method with electrospray ionization has been developed for analysis of mirtazapine in rat plasma. After addition of diazepam as internal standard, liquid–liquid extraction was used to produce a protein-free extract. Chromatographic separation was achieved on a 150 × 4.6 mm, 5 μm particle, ODS column with 84:16 (v/v) methanol–water containing 0.1% ammonium acetate and 0.01% glacial acetic acid as mobile phase. LC–MS–MS was performed in selected-ion-monitoring (SIM) mode using target fragment ions m/z 195.09 for mirtazapine and m/z 192.80 for the IS. Calibration plots were linear over the range of 0.516–618.8 ng mL^?1. The lower limit of quantification was 0.516 ng mL^?1. Intra-day and inter-day precision were better than 12.6 and 8.8%, respectively. Mean recovery of mirtazapine from plasma was in the range 87.41–90.06%; average recovery was 88.40% (RSD 3.95%). Significant gender differences between mirtazapine pharmacokinetic data were observed in this study.