LC APCI MS Research Articles

Identification of a novel component leading to anti-tumor activity besides the major ingredient cordycepin in Cordyceps militaris extract.

In accordance with our previous study that was carried out to identify novel anti-tumor ingredients, chromatographic separation in combination with an anti-tumor activity assay was used for analysis of Cordyceps militaris extract in this study. Various modes of chromatography including reversed-phase, cation-exchange and anion-exchange were used to separate components of Cordyceps militaris, which showed various chemical properties. Anti-tumor activity of each fraction was assessed by a Hoechst staining-based apoptosis assay using malignant melanoma MeWo cells. By these repeated approaches through chromatographic segregation and cell biological assay, we finally succeeded in identifying the target substance from a certain fraction that included neutral hydrophilic components using a pre-column and post-column chlorine adduct ionization LC-APCI-MS method. The target substance was a mono-carbohydrate, xylitol, that induced apoptotic cell death in MeWo cells but not in normal human OUMS-24 fibroblasts. This is the first study showing that Cordyceps militaris extract contains a large amount of xylitol. Thus, our results will contribute greatly to uncovering the mysterious multifunctional herbal drug Cordyceps militaris as an anti-tumor agent.

Development and validation of an LC–APCI-MS method for the determination of bisphenol A in hair samples of Greek population

Development and validation of an LC–APCI-MS method for the determination of bisphenol A in hair samples of Greek population

Imidacloprid and 6-chloronicotinic acid determination in blood serum of long-term exposed rabbits using LC–APCI-MS technique

Imidacloprid and 6-chloronicotinic acid determination in blood serum of long-term exposed rabbits using LC–APCI-MS technique

Quantification of phylloquinone and menaquinones in feces, serum, and food by high-performance liquid chromatography-mass spectrometry.

Vitamin K, comprising phylloquinone (PK) and menaquinones (MKn), is a family of vitamers found in multiple biological and environmental matrices. Advancing emerging evidence for novel and distinct physiologic roles of these vitamers in human health and disease necessitates sensitive and selective methods for quantifying PK and MKn in these matrices. We developed a novel method employing high-performance liquid chromatography-mass spectrometry with atmospheric pressure chemical ionization (LC-APCI-MS) for simultaneous quantification of 11 vitamin K vitamers that can be applied in feces, serum and food. Minimal detectable concentrations of vitamin K vitamers ranged from 1 pmol/g to 30 pmol/g. Limits of quantification ranged from 5 pmol/g to 90 pmol/g. Inter-assay and intra-assay variations were <17% and <8%, respectively, in food, and <12% and <8%, respectively, in feces. Recovery exceeded 80% for all vitamers in both food and feces. The method successfully quantified PK and MKn concentrations in rat chow, feces and serum. In summary, this LC-APCI-MS method provides a sensitive and selective tool for quantifying vitamin K vitamers in feces, serum and food. This method can be applied in human and animal studies examining the role of vitamin K vitamers derived from the diet and gut bacteria synthesis in health and disease.

Accurate quantitation of pentaerythritol tetranitrate and its degradation products using liquid chromatography–atmospheric pressure chemical ionization–mass spectrometry

After an explosion of pentaerythritol tetranitrate (PETN), its degradation products pentaerythritol trinitrate (PETriN), dinitrate (PEDiN) and mononitrate (PEMN) were detected using liquid chromatography–atmospheric-pressure chemical-ionization–mass spectrometry (LC–APCI–MS). Discrimination between post-explosion and naturally degraded PETN could be achieved based on the relative amounts of the degradation products. This information can be used as evidence when investigating a possible relationship between a suspect and a post-explosion crime scene. The present work focuses on accurate quantitation of PETN and its degradation products, using PETriN, PEDiN and PEMN standards specifically synthesized for this purpose. With the use of these standards, the ionization behavior of these compounds was studied, and a quantitative method was developed. Quantitation of PETN and trace levels of its degradation products was shown to be possible with accuracy between 85.7% and 103.7% and a precision ranging from 1.3% to 11.5%. The custom-made standards resulted in a more robust and reliable method to discriminate between post-explosion and naturally-degraded PETN.

Carotenoid content in vegetative and reproductive parts of commercially grown Moringa oleifera Lam. cultivars from India by LC–APCI–MS

Non-availability of standards is the main problem in quantification of carotenoids from plants. In this regard, six major carotenoids, i.e. all-E-luteoxanthin, 13-Z-lutein, all-E-lutein, all-E-zeaxanthin, 15-Z-β-carotene and all-E-β-carotene, were purified using open-column chromatography and thin-layer chromatography. Using these purified standards, carotenoids from fresh leaves, flowers and fruits of eight commercially grown cultivars of Moringa oleifera were quantified by liquid chromatography–mass spectrometry. All-E-lutein was found as the major carotenoid in leaves and fruits and accounted for 53.6 and 52.0 % of the total carotenoids, respectively. Among the eight cultivars screened, the cultivar Bhagya (KDM-1) had the maximum amount of all-E-zeaxanthin, all-E-β-carotene and total carotenoids. The methodology used in the present investigation for purification and quantification of carotenoids is simplified and hence finds use in screening of carotenoids in other plants. Results also explore the M. oleifera leaves as a rich source of carotenoids, which is significant for its implications in malnutrition programme to alleviate the vitamin A deficiency.

CHEMOMETRIC APPROACHES FOR THE CHARACTERIZATION OF TURKISH RHODODENDRON AND HONEYDEW HONEYS DEPENDING ON AMINO ACID COMPOSITION

The amino acid composition of twenty-five honey samples obtained from different regions of Turkey (rhododendron and honeydew honey) was determined by a liquid chromatography–atmospheric pressure chemical ionization–mass spectrometry (LC–APCI–MS). Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were used to characterize and classify the honey samples. In rhododendron honeys, major amino acids were detected as aspartic acid, lysine, and arginine, for honeydew honeys, lysine, arginine, and histidine were found, following the proline. Five principal components (PCs) and four main clusters were obtained from the chemometric analysis that classifies the samples. Five PCs were detected to be explanatory of more than 79% of the total variance in the data matrix. It can be concluded that free amino acids are good indicators for determination of botanical origins and chemometric studies are highly useful for honey characterization.

Development and application of LC–APCI–MS method for biomonitoring of animal and human exposure to imidacloprid

Imidacloprid (IMI) is a relatively new neuro-active neonicotinoid insecticide and nowadays one of the largest selling insecticides worldwide. In the present study a LC–APCI–MS based method was developed and validated for the quantification of imidacloprid and its main metabolite 6-chloronicotinic acid (6-CINA) in urine and hair specimens. The method was tested in biomonitoring of intentionally exposed animals and subsequently applied for biomonitoring of Cretan urban and rural population.The developed analytical method comprises two main steps of analytes isolation from specimen (solid–liquid extraction with methanol for hair, liquid–liquid extraction with methanol for urine) and subsequent instrumental analysis by LC–APCI–MS.The developed method was applied for the monitoring of IMI and 6-ClNA in hair and urine of laboratory animals (rabbits) intentionally fed with insecticide at low or high doses (40 and 80mgkg−1weightd−1 respectively) for 24weeks. The analytes were detected in the regularly acquired hair and urine specimens and their found levels were proportional to the feeding dose and time of exposure with the exception of slight decline of IMI levels in high dose fed rabbits after 24weeks of feeding. This decline can be explained by the induction of IMI metabolizing enzymes by the substrate. After testing on animal models the method was applied for pilot biomonitoring of Crete urban (n=26) and rural (n=32) population. Rural but not urban population is exposed to IMI with 21 positive samples (65.6%) and found median concentration 0.03ngmg−1. Maximum concentration detected was 27ngmg−1.

A new method for detection of five alternaria toxins in food matrices based on LC–APCI-MS

A new method for the detection of alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT), tentoxin (TEN), and tenuazonic acid (TeA), five alternaria toxins (ATs) was developed by liquid chromatography–triple quadrupole mass spectrometry equipped with atmospheric pressure chemical ionisation (APCI). A single extraction was used to recover the five ATs by apple juices, beers, tomato sauces, olives and dried basil. Different Solid Phase Extractions (SPE) and clean-up were selected to optimise the purification step for each food matrix. Limits of detection and quantification were, respectively, in the range 0.16–12.31 and 0.54–41.04ngg−1. Recovery rates were generally above 70%, except for dried basil and olives. Thirty out of 70 samples analysed (7 apple juices, 14 beers and 9 tomato sauces) resulted positive to at least one alternaria toxin investigated. AOH was the most common AT (14 samples), followed by ALT (10 samples). The highest concentration of ATs was found in commercial apple juices (35.33ngg−1).

Simultaneous pharmacokinetic assessment of cefadroxil and clavulanic acid in human plasma by LC–MS and its application to bioequivalence studies

A simple, rapid and selective liquid chromatography–atmospheric pressure chemical ionization–mass spectrometry (LC–APCI–MS) assay method has been developed and fully validated for the simultaneous quantification of cefadroxil (CF) and clavulanic acid (CA) in human plasma. Analytes and internal standard (IS) were extracted from human plasma by solid-phase extraction (SPE) technique using Sam prep (3mL, 100mg) extraction cartridge. The extracted samples were chromatographed on a reverse phase C18 column using a mixture of methanol: acetonitrile: 2mM ammonium acetate (pH 3.5) (25:25:50, v/v/v) as the mobile phase at a flow rate of 0.8mL/min. Quantification of the analytes and IS were carried out using single quadrupole LC–APCI–MS through selected-ion monitoring (SIM) at m/z 362 and m/z 198, for CF and CA, respectively. Method validation was performed as per the FDA guidelines and the results met the acceptance criteria. Plasma concentration of CF and CA followed by the oral administration of CF/CA (500/125mg) pill to healthy male volunteers (n=12) was measured. Area under plasma concentration–time curve from 0 to 12h (AUC0–12h) and 0h extrapolated to infinity (AUC0−∞) were calculated. The ratio of AUC0–12h/AUC0−∞ was found to be >85% for all the subjects, as recommended by the FDA guidelines.

Comparison of toxicokinetic and tissue distribution of triptolide-loaded solid lipid nanoparticles vs free triptolide in rats

The traditional Chinese medicine Tripterygium wilfordii Hook F (TWHF) is used clinically to treat some autoimmune and inflammatory disorders including rheumatoid arthritis, systemic lupus erythematosus, and skin diseases. However TWHF has a high potential for toxicity, so its clinical use is limited. Solid lipid nanoparticle (SLN) delivery systems are reported to have remarkable advantages over conventional formulations of bioactive plant extracts, such as enhancing solubility and bioavailability, offering protection from toxicity, and enhancing pharmacological activity. We reported previously that a tripterygium glycoside (TG) solid lipid nanoparticle (TG-SLN) delivery system had a protective effect against TG-induced male reproductive toxicity. To better understand this issue, we used triptolide (TP) as a model drug in a comparative study of the toxicokinetic and tissue distribution of TP-SLN and free TP in rats, allowing us to observing the in vivo behavior of this nanoformulation and to assess mechanisms of SLN-related toxicity. A fast and sensitive HPLC–APCI–MS/MS method was developed for the determination of triptolide in rat plasma. Fourteen rats were divided randomly into two groups of 7 rats each for toxicokinetic analysis, with one group receiving free TP (450μg/kg) and the other receiving the TP-SLN formulation (450μg/kg). Blood was obtained before dosing and 0.083, 0.17, 0.25, 0.33, 0.5, 0.75, 1, 1.5, 2, 3 and 4h after drug administration. Thirty-six rats were divided randomly into six equal groups for a tissue-distribution study. Half of the rats received intragastric administration of TP (450μg/kg) and the other half received TP-SLN (450μg/kg). At 15, 45, and 90min after dosing, samples of blood, liver, kidney, spleen, lung, and testicular tissue were taken. TP concentration in the samples was determined by LC–APCI–MS–MS. The toxicokinetic results for the nanoformulation showed a significant increase the area under the curve (AUC) (P<0.05), significantly longer Tmax and mean retention times (MRTs) (0–t) (P<0.05), significantly decreased Cmax (P<0.05). The nanoformulation promoted absorption with a slow release character, indicating that toxicokinetic changes may be the most important mechanism for the enhanced efficacy of nanoformulations. Tissue-distribution results suggest a tendency for TP concentrations in the lung and spleen to increase, while TP concentrations in plasma, liver, kidney, and testes tended to decrease in the TP-SLN group. At multiple time points, testicular tissue TP concentrations were lower in the TP-SLN group than in free TP group. This provides an important clue for the decreased reproductive toxicity observed with TP-SLN.

Highly sensitive, quick and simple quantification method for mono and disaccharides in aqueous media using liquid chromatography–atmospheric pressure chemical ionization–mass spectrometry (LC–APCI–MS)

A highly sensitive, rapid LC–APCI–MS method for identification and quantification of mono and disaccharides in simple or complex aqueous phase has been developed. This original method is easy to use, no derivation and no post-column injection are needed. The separation is performed with a hydrophilic amino interaction (HILIC) column allowing high-throughput analysis with analysis times of 15 min for monosaccharides to 22 min for disaccharides. The development of the method carried out with 9 standard saccharides allowed to point out a dynamic range from 0.1–25.6 to 1–256 μg mL −1 depending on the considered sugar. Next, the method was validated on saccharides at known concentrations in water and on 2 real samples: orange juice and aqueous phase obtained after enzymatic hydrolysis of sunflower seeds.

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.

Determination of Pilocarpine in Human Plasma by LC–APCI–MS–MS and Application to a Pharmacokinetic Study

A sensitive, specific and rapid high performance liquid chromatography–atmospheric pressure chemical ionization source-tandem mass spectrometry (LC-APCI-MS-MS) method for the determination of pilocarpine in human plasma was developed and validated. The method is based on liquid–liquid extraction, followed by a reversed-phase liquid chromatographic separation, and detected by means of tandem mass spectrometry. The linear calibration curve covered a concentration range of 2–500 μg L−1. The intra- and inter-day precisions for pilocarpine were <10% and the accuracies were between 90 and 110%. The method was applied successfully to a pharmacokinetic study involving 20 healthy Chinese male volunteers after oral administration of 6 mg pilocarpine.

LC–APCI–MS Quantitative Analysis of Curcumin in Rabbit Plasma

A simple and specific liquid chromatography–atmospheric pressure chemical ionization–mass spectrometry (LC–APCI–MS) method was developed and validated for quantitative analysis of curcumin in rabbit plasma. Chromatographic separation was carried out on Zorbax SB-C18 column, acetonitrile-methanol–water used as mobile phase with gradient elution. APCI source was applied and operated in negative ion mode, and selective ion monitoring mode used to quantify curcumin. The method showed excellent intra-assay and inter-assay precision (relative standard deviations and bias <15%) for quality control (QC) samples. This method was successfully applied to a pharmacokinetic study of curcumin after intravenous administration to rabbits.

Vitamin D3 quantification in a cod liver oil-based supplement

A reliable, accurate and reproducible method to quantify vitamin D3 (Vit. D3) in oily dietary supplements was developed after three Vit. D3 intoxications were diagnosed as reasonably resulting from a dietary administration of a cod liver oil based supplement. Liquid chromatography coupled to mass spectrometry operating in atmospheric pressure chemical ionization conditions (LC–APCI) and by using a deuterium labelled internal standard resulted to be an effective technique to reach the analytical aim. Due to the complexity of the oily matrix, the new analytical approach required a solid phase extraction step prior to analysis. The amount of Vit. D3 declared on the label of the cod liver oil based supplement for each soft capsule is 1.5 μg. Consequently, the method was developed to quantify Vit. D3 amounts in the range 1–5 μg/mL. To improve reliability of obtained data, both MS and MS/MS acquisition methods were employed. The method was evaluated by measuring the characteristic parameters such as linearity, precision, accuracy and robustness and cross checked against a certified pharmaceutical preparation. The LC–APCI–MS and MS/MS methods were applied in order to assess the Vit. D3 content in the dietary supplements taken by the intoxicated patients, found about three order of magnitude higher than that declared. The Vit. D3 content of other batches of the same commercial product was found as declared.

LC–APCI–MS Simultaneous Determination of Barbital, Amobarbital, Phenobarbital and Secobarbital in Human Plasma

A sensitive and simple liquid chromatography–atmospheric pressure chemical ionization–mass spectrometry (LC–APCI–MS) method was developed and validated for determination of barbital, amobarbital, phenobarbital and secobarbital in human plasma. Barbiturate drugs extracted from plasma samples using ethyl ether and separation were carried out on an Agilent Zorbax SB-C18 column (2.1 mm × 150 mm, 5 μm) at 30 °C, acetonitrile–water used as mobile phase with gradient elution. APCI source was applied and operated in negative ion mode, and selective ion monitoring mode used to quantify barbiturate drugs. The method showed excellent intra-assay and inter-assay precision (relative standard deviation and bias <15%) for quality control samples spiked at concentrations of 10, 400 and 1,600 ng mL−1 and the r 2 > 0.99 over the range were investigated (10–2,000 ng mL−1). Lower limits of quantification were estimated to be 10 ng mL−1. This method is simple and sensitive for quantitation of barbiturate drugs in plasma, suitable for use in the clinical and forensic medical laboratory.

LC–APCI-MS–MS Analysis of Zidovudine in Human Plasma, and Application to a Bioequivalence Study

The objective of this work was to establish a sensitive, simple, accurate, and reliable LC–APCI-MS–MS method for analysis of zidovudine in human plasma. Plasma samples were processed by protein precipitation with perchloric acid solution. Separation on a 250 mm × 4.6 mm, 5 μm particle, C18 column was achieved by use of methanol–water 65:35 (v/v) containing 0.2% formic acid as mobile phase. Mass spectrometric quantification was performed in positive-ion multiple reaction monitoring (MRM) mode, by monitoring the m/z transitions 268.1 → 127.0 for zidovudine and m/z 248.1 → 121.0 for tinidazole (internal standard). The calibration plot was linear in the concentration range 1.638–6250 μg L−1, and the lower limit of quantification (LLOQ) was 1.638 μg L−1. Intra-day and inter-day precision (as RSD) for zidovudine at four quality-control levels was less than 15%, and accuracy was between 95.1 and 114.2%. The method enables simple, sensitive, and accurate analysis of zidovudine in human plasma, and was successfully used in a bioequivalence study of 48 healthy Chinese volunteers.

Quantitation of ceramides in nude mouse skin by normal-phase liquid chromatography and atmospheric pressure chemical ionization mass spectrometry

A sensitive and accurate normal-phase liquid chromatography and atmospheric pressure chemical ionization mass spectrometry (LC–APCI–MS) method for determining the standard ceramide [NS] (Cer[NS]) was developed and validated so as to improve the traditional thin-layer chromatography (TLC) technique and LC–electrospray ionization (ESI)–MS method to profile and quantify ceramides in nude mouse skin. Normal-phase LC–APCI–MS was optimized to separate the nine classes of ceramides presented in the stratum corneum (SC) of nude mouse skin. A normal-phase silica column eluted with the gradient system from heptane:acetone/butanol (90:10, v/v) of 75:25 to 100% acetone/butanol (90:10, v/v) (with each solvent containing 0.1% [v/v] triethylamine and 0.1% [v/v] formic acid) at a flow rate of 0.8 ml/min was found to be optimal for analyzing standard Cer[NS]. The analysis of Cer[NS] was validated and employed as the standard for constructing a calibration curve to quantitate all classes of ceramides. This method was applied to profile the classes and contents of ceramides in the SC of nude mouse skin and proved to be workable. It was concluded that this improved method can be used to directly detect and quantify all classes of ceramides in the SC of nude mouse skin and that it is more convenient and labor-saving than the traditional TLC method.

Detection of macrocyclic lactones in porcine liver, meat and fish tissue using LC–APCI–MS–MS

A selective and sensitive method for the simultaneous determination of five avermectins (abamectin, ivermectin, doramectin, emamectin and eprinomectin) and one milbemycin (moxidectin) in porcine liver, bovine meat and fish tissue was developed. The method involved extraction with acetonitrile and purification by C18 solid-phase extraction. Detection was carried out using liquid chromatography coupled to multiple mass spectrometry (LC–MS2) equipped with APCI in the negative mode. This method was validated according to the requirements of Commission Decision EC/2002/657 (Implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. Off J Eur Commun. L221: 8–36.). In addition to the linear response (R 2 between 59 and 97%), good repeatability (CV between 20 and 35%), reproducibility (CV between 20 and 35%) and detection (CCα) and quantification (CCβ) limits were obtained for all compounds in all matrices considered.