Heated Electrospray Ionization Tandem Mass Research Articles

Development of a novel method for multiple phytohormone analysis by UHPLC-MS/MS from bio-enriched organic fertilizer prepared using banana pseudostem sap waste.

Banana harvesting generates a large amount of banana pseudostem waste, which is generally burnt or thrown away, despite containing many nutrients. Bio-enriched organic fertilizer (BOF) was prepared from banana pseudostem sap (BPS), and it has been patented (Patent No. WO 2013/001478 Al). Several reports revealed that its application increases plant growth promotion of various horticulture crops. Apart from macro- and micronutrients, it also contained phytohormones. Hence, the present study aims to detect and quantify phytohormone in it. A novel method was developed to extract four phytohormones, viz., indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), gibberellic acid (GA3), and salicylic acid (SA) using single solvent from BPS and BOF. Extracted hormones were analyzed by ultrahigh-performance liquid chromatography coupled with heated electrospray ionization tandem mass spectrometry (UHPLC-HESI-MS/MS). BOF showed a higher concentration of IAA, IBA, GA3, and SA than BPS. Thus, this is the first time a method has been reported to extract and detect phytohormones from banana pseudostem sap.

UHPLC-MS/MS and QRT-PCR profiling of PGP agents and Rhizobium spp. of induced phytohormones for growth promotion in mungbean (var. Co4)

In present study, five potential strains with different plant growth promotion (PGP) characteristics were used. By considering various PGP properties of different bacterial strains, several treatments based on various combinations were developed and studied on mungbean (var. Co4). The quantification of the phytohormones was performed on ultrahigh-performance liquid chromatograph coupled to heated electrospray ionization tandem mass spectrometry (UHPLC/HESI-MS/MS). Indole 3-acetic acid (IAA) and Indole 3-butyric acid (IBA) were quantified in positive ionization mode while Gibberellic acid (GA3) and salicylic acid (SA) were quantified in negative ionization mode. Among all the treatments two penta combinations of consortia 1 (Rhizobium + Azospirillum + Pseudomonas + Bacillus spp. + Bacillus licheniformis) and consortia 2 (Rhizobium + Azotobacter + Pseudomonas + Bacillus spp. + Bacillus licheniformis) were found most effective. Higher amount of IAA (1.043 μg g−1), IBA (0.036 μg g−1), GA3 (1.999 μg g−1) and SA (0.098 μg g−1) Fresh weight (FW) were found in treated adolescent root tissues of consortia 2 as compared to consortia 1. Moreover, transcriptional level of the plant hormones were 2–4 fold higher in the relative gene expression study of three genes: ARF (Auxin responsive factors), ERF-IF (Ethylene-responsive Initiation Factors) and GAI (Gibberellic-Acid Insensitive) in consortia 2, on the 15th, 30th and 45th day using quantitative real time-Polymerase chain reaction (qRT-PCR). Furthermore, Yield attributing characters like, the number of nodules plant−1, number of pods plant−1, weight of nodule and seed yield plant−1 were also increased as compared to the control. As a result, the current research elucidated that penta combinations consortium of Rhizobium sp. and rhizobacteria can be developed as a single delivery system biofertilizer for enhancing mungbean productivity.

Are Rubber Gloves Marketed as Accelerator-Free Truly Free of Accelerators?

Allergic contact dermatitis to rubber accelerators in gloves has been well described in the literature. In response to this, glove manufacturers have recently marketed "accelerator-free" gloves. Little research has been done, to confirm whether these gloves are truly free from the accelerators known to cause contact dermatitis. The aim of the study was to verify use of accelerators in reportedly accelerator-free/low-dermatitis-potential gloves. A total of 16 commercially available medical gloves touted as "accelerator-free," "sensitive," or "low dermatitis potential" were obtained and analyzed via mass spectrometry (liquid chromatography heated electrospray ionization tandem mass spectrometry and liquid chromatography heated electrospray high-resolution tandem mass spectrometry) to determine whether any of the 9 known rubber accelerators were present (thiurams, carbamates, mercaptobenzothiazole, and diphenylguanidine). Despite marketing claims to the contrary, all tested gloves had at least 1 accelerant detected. Dipentamethylenethiuram disulfide, a thiuram, was found in all 16 gloves. Half of the gloves (8/16) contained more than 1 accelerator, with 1 glove having 5 rubber accelerators present. Patients with allergic contact dermatitis to accelerators should be aware potentially sensitizing accelerators may be present in gloves that are reported to not contain them.

Development and validation of an absolute protein assay for the simultaneous quantification of fourteen CYP450s in human microsomes by HPLC-MS/MS-based targeted proteomics

The cytochrome P450 (CYP450) superfamily constitutes the major enzymatic system involved in drug metabolism. CYP450s are highly expressed in the liver and other tissues and limited data on absolute characterization of CYP450s in extra hepatic organs, such as the small intestine, are available. Our objective was to develop and validate an absolute quantification assay by HPLC-MS/MS-based targeted proteomics allowing the simultaneous quantification of fourteen major human CYP450 isoforms (CYP1A1, 1A2, 1B1, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2, 3A4, 3A5, 3A7 and 4F2) in human liver and intestine microsomes. Absolute protein quantification was performed using two proteotypic peptides for each of the fourteen CYP450s. Peptides were obtained after a tryptic digestion of microsomes and samples were analyzed by high performance liquid chromatography with heated electrospray ionization tandem mass spectrometry (HPLC-HESI-MS/MS). Chromatographic separation was performed on a Biobasic-8 analytical column (5 μm 100 x 1 mm) with a gradient elution using acetonitrile and water both fortified with 0.1% formic acid (flow rate: 75 μL/min). Calibration curves were linear over a wide range of concentrations (0.1–50 nM) and the assay met all requirements of sensitivity, linearity, precision, accuracy and matrix effect. Strong correlations were observed between the two proteotypic peptides for each isoenzyme, corroborating the strength of this method. Twelve CYP450s were detected in commercially available human liver microsomes while seven CYP450s were detected in human intestine microsomes. To our knowledge, this is the most sensitive (0.1 nM) and the first most extensively validated assay that can be applied to the absolute quantification of CYP450s in human tissues.

Highly sensitive LC-MS/MS methods for the determination of seven human CYP450 activities using small oral doses of probe-drugs in human.

Cocktails composed of several Cytochrome P450 (CYP450)-selective probe drugs have been shown of value to characterize in vivo drug-metabolism activities. Our objective was to develop and validate highly sensitive and selective LC-MS/MS assays allowing the determination of seven major human CYP450 isoenzyme activities following administration of low oral doses of a modified CYP450 probe-drug cocktail in patients. The seven-drug cocktail was composed of caffeine, bupropion, tolbutamide, omeprazole, dextromethorphan, midazolam (all administered concomitantly) and chlorzoxazone (administered separately) to phenotype for CYP1A2, 2B6, 2C9, 2C19, 2D6, 3A4/5 and 2E1, respectively. Serial plasma and urine samples were collected over an 8h period. The probe-drugs and their respective metabolites were measured in both human plasma and urine, except for omeprazole (plasma only) and chlorzoxazone (urine only). Samples were analyzed by high performance liquid chromatography with heated electrospray ionization tandem mass spectrometry (HPLC-HESI-MS/MS) using a Phenomenex Luna PFP (2) analytical column (3μm PFP(2) 150×3mm) for chromatographic separation. Optimal detection was achieved based on 3 different analytical methods; (1) isocratic elution with a mobile phase consisting of acetonitrile and water both fortified with 0.01% formic acid for the analysis of bupropion, tolbutamide, chlorzoxazone and their respective metabolites; (2) isocratic elution with a mobile phase composed of acetonitrile and ammonium formate (pH 3; 10mM) for omeprazole, dextromethorphan, midazolam and their metabolites; (3) for caffeine and paraxanthine, gradient elution using acetonitrile and 0.01% formic acid in water was used. All calibration functions were linear for all probe drugs and metabolites in both matrices over wide analytical ranges. The main advantages of our methods are the use of specific probe drugs available in most countries, the administration of small doses of probe drugs, small volume of plasma required for the analyses and simple and rapid extraction procedures. The methods met all requirements of specificity, sensitivity, linearity, precision and accuracy and stability generally accepted in bioanalytical chemistry. Determination of CYP450 phenotype in patients will permit characterization of their capacities to metabolize drugs through CYP450 under specific conditions at a definite time. This tool will be highly clinically relevant since wide intersubject variability observed in drug response is largely explained by variation in drug metabolism; it will be particularly useful in polymedicated patients with multiple comorbidities. So far, our CYP450 cocktail assays have been successfully applied to phenotype CYP450 activities in patients.

Simultaneous determination of ten anticoagulant rodenticides in tissues by column-switching UHPLC-ESI-MS/MS.

This paper describes the development of a method for the simultaneous determination of ten anticoagulant rodenticides (coumafuryl, warfarin, pindone, coumatetralyl, coumachlor, difenacoum, bromadiolone, brodifacoum, chlorophacinone and flocoumafen) in the liver and kidney based on column-switching liquid chromatography coupled with heated electrospray ionization tandem mass spectrometry. The simple sample preparation includes extraction with methanol. A C18 trapping column was used for online solid-phase extraction before analytical separation with the mobile phase comprising a mixture of 0.1% formic acid in water, methanol and acetonitrile. Chromatographic separation was achieved using a Thermo Hypersil ultra high-performance liquid chromatography (UHPLC) C18 column with the mobile phase consisting of 5 mM ammonium formate buffer (pH = 9) and methanol. The column-switching procedure ensured no matrix effects during electrospray ionization (ESI). Extraction recoveries ranged between 91 and 100% for liver and between 89 and 97% for kidney. The method showed good linearity up to 750 ng g(-1). The limit of detection ranged between 0.001 and 0.022 ng g(-1) for liver and between 0.001 and 0.028 ng g(-1) for kidney. The developed method was successfully used in several animal poisoning cases.

Development and validation of a liquid chromatography–tandem mass spectrometry method for the quantitative determination of gamithromycin in animal plasma, lung tissue and pulmonary epithelial lining fluid

A sensitive and specific method for the quantitative determination of gamithromycin in animal plasma, lung tissue and pulmonary epithelial lining fluid (PELF) using liquid chromatography combined with heated electrospray ionization tandem mass spectrometry (LC–MS/MS) was developed. The sample preparation was rapid, straightforward and consisted of a deproteinization and phospholipid removal step using an Oasis® Ostro™ 96-well plate (chicken, turkey and calf plasma) or HybridSPE®-Phospholipid SPE cartridges (pig plasma and turkey lung tissue), while a liquid–liquid extraction with diethyl ether in alkaline medium was used for PELF of turkey poults. Chromatography was performed on a C18 Hypersil GOLD column using 0.01M ammonium acetate in water with a pH of 9, and acetonitrile as mobile phases. The MS/MS instrument was operated in the positive electrospray ionization mode and the following selected reaction monitoring transitions were monitored for gamithromycin (protonated molecule>product ion): m/z 777.45>619.35 and m/z 777.45>157.80 for quantification and identification, respectively. The method was validated in-house: matrix-matched calibration graphs were prepared and good linearity (r≥0.99) was achieved over the concentration ranges tested (2.5–10,000ngmL−1 for chicken, pig and calf plasma; 5.0–2500ngmL−1 for turkey plasma; 50–10,000ngg−1 for turkey lung tissue and 20–1000ngmL−1 for turkey PELF). Limits of quantification (LOQ) were 2.5ngmL−1 for chicken, pig and calf plasma and 5.0ngmL−1 for turkey plasma, while the limits of detection (LOD) ranged between 0.007 and 0.07ngmL−1. For lung tissue and PELF, respective LOQ and LOD values of 50ngg−1 and 0.76ngg−1 (lung tissue) and 20ngmL−1 and 0.1ngmL−1 (PELF) were obtained. The results for the within-day and between-day precision, expressed as relative standard deviation (RSD), fell within the maximal RSD values. The accuracy fell within −30% to +10% (concentrations 1–10ngmL−1) or −20% to +10% (concentrations>10ngmL−1 or ngg−1) of the theoretical concentration.The method was successfully applied for the quantitative determination of gamithromycin in plasma samples of chickens, turkeys, pigs and calves; and in lung tissues and PELF of turkeys, all derived from pharmacokinetic studies in these animal species.

Simultaneous quantification of idelalisib, fludarabine and lenalidomide in rat plasma by using high-performance liquid chromatography coupled with heated electrospray ionization tandem mass spectrometry

A sensitive and reliable high-performance liquid chromatography-mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous quantification of idelalisib, fludarabine and lenalidomide using tolbutamide as an internal standard. Analytes were recovered by liquid-liquid extraction and separated on a reverse phase C18 column (150mm×4.6mm i.d., 5μm) using methanol:0.1% formic acid buffer (70:30) as mobile phase at a flow rate of 1mL/min in isocratic mode. Selective reaction monitoring was performed using the transitions, i.e. m/z 416.25/176.48, 286.11/154.10, 260.15/149.15, and 271.14/155.06 to quantify idelalisib, fludarabine and lenalidomide and tolbutamide, respectively. The method was validated over the concentration range of 1.15-576.84ng/mL for idelalisib, 0.95-476.25ng/mL for fludarabine and 0.97-486.19ng/mL for lenalidomide. Intra and inter-day accuracy and precision of validated method were within the acceptable limits of <15%. Coefficients of correlation (r(2)) for the calibration curves were >0.998 for all analytes. The method was successfully applied for simultaneous estimation of idelalisib, fludarabine and lenalidomide in a pharmacokinetic study in rats.

Quantitative determination of the Fusarium mycotoxins beauvericin, enniatin A, A1, B and B1 in pig plasma using high performance liquid chromatography–tandem mass spectrometry

A sensitive and reliable method was developed for the identification and quantification of beauvericin, enniatin A, A1, B and B1 in pig plasma using liquid chromatography combined with heated electrospray ionization tandem mass spectrometry. Sample clean-up consisted of a deproteinization step using acetonitrile, followed by evaporation of the supernatant and resuspension of the dry residue in acetonitrile/water (80/20, v/v). The method was in-house validated: matrix-matched calibration graphs were prepared for all compounds and correlation and goodness-of-fit coefficients ranged between 0.9980 and 0.9995 and between 5.2% and 11.3%, respectively. The within- and between-run precision and accuracy were evaluated and the results fell within the ranges specified. The limits of quantification were 0.1ng/mL for enniatin A and A1 and 0.2ng/mL for beauvericin, enniatin B and B1, whereas limits of detection were≤10pg/mL for all analytes. The method has been applied for the analysis of real plasma samples from one pig that received an oral bolus (0.05mg/kg BW) of the investigated mycotoxins. At the applied dosage, the results indicated the suitability of the method for use in toxicokinetic studies with enniatins.

Simultaneous analysis of chlorpyrifos and cypermethrin in cord blood plasma by online solid-phase extraction coupled with liquid chromatography–heated electrospray ionization tandem mass spectrometry

Chlorpyrifos and cypermethrin are the most used insecticides in Taiwan. Exposure to both pesticides has been associated with reproductive and developmental health effects in humans and animals. This study describes an online solid-phase extraction coupled with liquid chromatography–heated electrospray ionization tandem mass spectrometry (online SPE-LC/HESI/MS/MS) method to analyze chlorpyrifos and cypermethrin in cord blood of pregnant women. Calibration curves showed good linearity ( r 2 > 0.998) for both pesticides within the range of 0.1–100 ppb. Limits of detection (LODs) were 0.01 and 0.05 ppb and recoveries in cord blood were 97.2 ± 4.8% and 93.5 ± 9.5% for chlorpyrifos and cypermethrin respectively. After analysis of 396 samples, the mean concentrations of chlorpyrifos and cypermethrin were 0.38 and 1.08 ppb respectively. These results demonstrate that LC/HESI/MS/MS is effective for the simultaneous analysis of chlorpyrifos and cypermethrin in cord blood with excellent sensitivity and specificity and may also be effective for high throughput assay in future epidemiology studies.

Development and validation of efficient stable isotope dilution LC–HESI–MS/MS method for the verification of β-lyase metabolites in human urine after sulfur mustard exposure

As a part of the project for screening unequivocal biomarkers after sulfur mustard exposure, a quantitative method for the determination of β-lyase metabolites 1,1′-sulfonylbis-[2-(methylsulfinyl)ethane] (SBMSE) and 1-methylsulfinyl-2-[2-(methylthio)ethylsulfonyl]ethane (MSMTESE) was validated. Full validation was conducted according to the FDA guidelines for method validation using pooled human urine as a sample matrix. The metabolites were extracted from urine with an optimized sample preparation procedure using ENV+ solid phase extraction cartridge with reduced volume of sample and solvents. Metabolites were detected by improved and faster liquid chromatography–heated electrospray ionization–tandem mass spectrometry (LC–HESI–MS/MS) method with two transitions of each chemical using non-buffered eluents, post-column splitter and higher flow-rate. These provided over five times faster analysis than previously published method providing 4.5 min/sample cycle time, to achieve up to 200 samples per day (24 h). Quantification was performed using deuterium labelled internal standard of SBMSE. The method was linear over the concentration range of 5–200 ng/ml (average correlation coefficients were R 2 = 0.997 and R 2 = 0.989) for both β-lyase metabolites, SBMSE and MSMTESE, respectively. The average retention times for SBMSE and MSMTESE were 1.96 ± 0.01 min and 3.24 ± 0.03 min ( n = 54). Calculated limits of detection were 4 ng/ml for both SBMSE and MSMTESE, respectively. Lower limits of quantification were 10 ng/ml and 11 ng/ml for SBMSE and MSMTESE, respectively. This validated method was successfully used in the First Confidence Building Exercise on Biomedical Samples Analysis organized by the Organisation for the Prohibition of Chemical Weapons (OPCW). Identification criteria for analysing unequivocal biomarkers of sulfur mustard with LC–MS/MS after alleged use is discussed and proposed based on the validation and exercise results.

The bioanalysis of vinorelbine and 4‐O‐deacetylvinorelbine in human and mouse plasma using high‐performance liquid chromatography coupled with heated electrospray ionization tandem mass–spectrometry

A sensitive, specific and efficient high-performance liquid chromatography/tandem mass spectrometry assay for the simultaneous determination of vinorelbine and its metabolite 4-O-deacetylvinorelbine in human and mouse plasma is presented. Heated electrospray ionization was applied followed by tandem mass spectrometry. A 50 microL plasma aliquot was protein precipitated with acetonitrile-methanol (1:1, v/v) containing the internal standard vinorelbine-d3 and 20 microL volumes were injected onto the HPLC system. Separation was achieved on a 50 x 2.1 mm i.d. Xbridge C(18) column using isocratic elution with 1 mm ammonium acetate-ammonia buffer pH 10.5-acetonitrile-methanol (28:12:60, v/v/v) at a flow rate of 0.4 mL/min. The HPLC run time was 5 min. The assay quantifies both vinorelbine and 4-O-deacetylvinorelbine from 0.1 to 100 ng/mL using sample volumes of only 50 microL. Mouse plasma samples can be quantified using calibration curves prepared in human plasma. Validation results demonstrate that vinorelbine and 4-O-deacetylvinorelbine can be accurately and precisely quantified in human and mouse plasma with the presented method. The assay is now in use to support (pre-)clinical pharmacologic studies with vinorelbine in humans and mice.

Determination of acyclovir in horse plasma and body fluids by high‐performance liquid chromatography combined with fluorescence detection and heated electrospray ionization tandem mass spectrometry

Two methods are presented for the determination of 'respectively' the plasma protein unbound and total concentration of acyclovir in horse plasma and body fluids: first, a liquid-liquid extraction was performed on plasma, combined with HPLC-fluorescence detection for the total plasma concentration; second a more sensitive method using high-performance liquid chromatography combined with heated electrospray ionization tandem mass spectrometry (LC-HESI-MS/MS) was described for plasma and for body fluids analysis. To obtain the unbound concentration of acyclovir in plasma, a simple deproteinization step using a Microcon filter was performed. Ganciclovir was used as an internal standard. Analysis was carried out on an Inertsil 5 ODS-3 column for the HPLC-fluorescence method. For the LC-HESI-MS/MS method a PLRP-S column was used. The limit of quantification (LOQ) for the total concentration was set at 50 and 2 ng mL(-1) for the HPLC-fluorescence method and the LC-HESI-MS/MS method, respectively. The limit of quantification for the unbound concentration was set at 5 ng mL(-1) and at 2 ng mL(-1) for body fluids. The methods were successfully used to perform pharmacokinetic and clinical studies in horses after intravenous and oral dosage of acyclovir and its prodrug valacyclovir.

Multi-residue analysis of eight anticoagulant rodenticides in animal plasma and liver using liquid chromatography combined with heated electrospray ionization tandem mass spectrometry

A sensitive method for the simultaneous quantification of eight anticoagulant rodenticides (brodifacoum, bromadiolone, chlorophacinone, coumatetralyl, difenacoum, difethialone, flocoumafen and warfarin) in animal plasma and liver using liquid chromatography combined with heated electrospray ionization tandem mass spectrometry (LC-HESI-MS/MS) is described. The sample preparation includes a liquid–liquid extraction with acetone. The compound 7-acetoxy-6-(2,3-dibromopropyl)-4,8-dimethylcoumarin is used as an internal standard. Chromatographic separation was achieved using a Nucleodur C18 gravity column. Good linearity was observed up to 750 ng mL −1 for chlorophacinone and up to 500 ng mL −1 for the other compounds in plasma. In liver, good linearity was seen up to 500 ng g −1 for brodifacoum, chlorophacinone, difenacoum and difethialone and up to 750 ng g −1 for the other compounds. Depending on the compound, a level of 1 or 5 ng mL −1 could be quantified fulfilling the criteria for accuracy and precision and was therefore set as limit of quantification of the method in plasma. In liver, the limit of quantification was set at 250 ng g −1 for coumatetralyl and warfarin and at 100 ng g −1 for the other compounds. In plasma, the limit of detection varied from 0.07 ng mL −1 for flocoumafen to 3.21 ng mL −1 for brodifacoum. In liver, the limit of detection varied from 0.37 ng g −1 for warfarin to 4.64 ng g −1 for chlorophacinone. The method was shown to be of use in a pharmacokinetic study after single oral administration to mice and in the confirmation of suspected poisoning cases in domestic animals.