Determination ofN,N-dimethyltryptamine inMimosa tenuiflorainner barks by matrix solid-phase dispersion procedure and GC-MS

Publication year: 2011Source: Journal of Chromatography B, Available online 15 November 2011Alain Gaujac, Adriano Aquino, Sandro Navickiene, Jailson Bittencourt de AndradeN,N-dimethyltryptamine (DMT) is a potent hallucinogen founded in beverages co…

Publication year: 2011
Source: Journal of Chromatography B, Available online 15 November 2011

Alain Gaujac, Adriano Aquino, Sandro Navickiene, Jailson Bittencourt de Andrade

N,N-dimethyltryptamine (DMT) is a potent hallucinogen founded in beverages consumed in religion rituals and neo-shamanic practices over the world. Two of these religions, Santo Daime and União do Vegetal (UDV), are represented in countries including Australia, the United States and several European nations. In some of this countries there have been legal disputes concerning the legalization of ayahuasca consumption during religious rituals, a beverage rich in DMT. In Brazil, even children and pregnant women are legally authorized to consume ayahuasca in religious context. A simple and low-cost method based on matrix solid-phase dispersion (MSPD) and gas chromatography with mass spectrometric detection (GC-MS) has been optimized for the determination ofN,N-dimethyltryptamine inM. tenuiflorainner bark. The experimental variables that affect the MSPD method, such as the amounts of solid-phase and herbal sample, solvent nature, eluate volume and NaOH concentration were optimized using an experimental design. The method showed good linearity (r = 0.9962) and repeatability (RSD < 7.4%) for DMT compound, with detection limit of 0.12 mg/g. The proposed method was used to analyze 24 samples taken from domestic locals. The results showed that concentrations of the target compound inM. tenuiflorabarks, ranged from 1.26 to 9.35 mg/g for these samples.

Highlights

► It is new the use of MSPD procedure and GC-MS method for the determination ofN,N-dimethyltryptamine inM. tenuiflorabarks; ► Use of an analytical standard of DMT isolated fromM. tenuiflora; ► The proposed method uses small amounts of sample and low demand for reagents and solvents.


LC-MS/MS-based metabolites ofEurycoma longifolia(Tongkat Ali) in Malaysia (Perak and Pahang)

Publication year: 2011Source: Journal of Chromatography B, Available online 9 November 2011Lee Suan Chua, Nor Amaiza Mohd Amin, Jason Chun Hong Neo, Ting Hun Lee, Chew Tin Lee, …A number of three LC-MS/MS hybrid systems (QTof, TripleTof and QTra…

Publication year: 2011
Source: Journal of Chromatography B, Available online 9 November 2011

Lee Suan Chua, Nor Amaiza Mohd Amin, Jason Chun Hong Neo, Ting Hun Lee, Chew Tin Lee, ...

A number of three LC-MS/MS hybrid systems (QTof, TripleTof and QTrap) has been used to profile small metabolites (m/z 100-1000) and to detect the targeted metabolites such as quassinoids, alkaloids, triterpene and biphenylneolignans from the aqueous extracts ofEurycoma longifolia.The metabolite profiles of small molecules showed four significant clusters in the principle component analysis for the aqueous extracts ofE. longifolia,which had been collected from different geographical terrains (Perak and Pahang) and processed at different extraction temperatures (35 °C and 100 °C). A small peptide of leucine (m/z 679) and a new hydroxyl methyl β-carboline propionic acid have been identified to differentiateE. longifoliaextracts that prepared at 35 °C and 100 °C, respectively. From the targeted metabolites identification, it was found that 3,4ɛ-dihydroeurycomanone (quassinoids) and eurylene (squalene-type triterpene) could only be detected in the Pahang extract, whereas canthin-6-one-3N-oxide could only be detected in the Perak extract. Overall, quassinoids were present in the highest concentration, particularly eurycomanone and its derivatives compared to the other groups of metabolites. However, the concentration of canthin-6-one and β-carboline alkaloids was significantly increased when the roots of the plant samples were extracted at 100 °C.

Highlights

E. longifoliahas different metabolite profiles dependent on the geographical origin. ► Leucine based peptide has been identified as marker in the room temperature extracts. ► A new hydroxyl methyl β-carboline propionic acid has been identified in the 100 °C extracts. ► Eurycomanone and its derivatives present in the highest amount compared to other groups. ► The concentration of alkaloids was higher than quassinoids in the 100 °C extracts.


Development and validation of a liquid chromatography–tandem mass spectrometry method for the quantification of opiorphin in human saliva

Publication year: 2011Source: Journal of Chromatography B, Available online 9 November 2011Lidija Brkljačić, Maja Sabalić, Ivan Salarić, Ivanka Jerić, Ivan Alajbeg, …Opiorphin, QRFSR-peptide, is a mature product of the PROL1 (proline rich, …

Publication year: 2011
Source: Journal of Chromatography B, Available online 9 November 2011

Lidija Brkljačić, Maja Sabalić, Ivan Salarić, Ivanka Jerić, Ivan Alajbeg, ...

Opiorphin, QRFSR-peptide, is a mature product of the PROL1 (proline rich, lacrimal 1) protein that showed beneficial effects in pain management, antidepressant-like actions as well as involvement in colonic motility and erectile physiology. Using opiorphin as a potential biomarker of different pathological states requires the development of robust and sensitive methods. We report a highly sensitive and specific liquid chromatography with tandem mass spectrometric detection (LC–MS/MS) analytical method for the analysis of opiorphin in human saliva. Quantification was based on multiple reaction monitoring using characteristic transitions (m/z347/120–as quantifying ion; 347/175 and 347/268 as qualifying ions). The assay was linear in the range of 0-110 ng/ml and the lower limit of quantification reached was 1.0 ng/ml. The intra-day precision and accuracy were between 2.7-5.6% and -2.3-3.2%, respectively. The inter-day precision and accuracy were between 10.8-13.7% and -11.0-52%, respectively. Mean recovery was 106% and mean matrix effect was 0.97. Opiorphin in TFA treated saliva samples was stable for at least 12 h at room temperature and up to 30 days at -20 °C. Opiorphin levels in human saliva samples collected from young healthy individuals ranged from 2.8 to 25.9 ng/ml.

Highlights

► Opiorphin, the QRFSR-pentapeptide, showed beneficial effects in pain management ► We developed a sensitive and specific liquid chromatography with tandem mass spectrometric detection (LC–MS/MS) analytical method for the analysis of opiorphin in human saliva. ► Opiorphin levels in human saliva samples collected from young healthy individuals ranged from 2.8 to 25.9 ng/ml.


Implementation of a cost-effective HPLC/UV-approach for medical routine quantification of donepezil in human serum

Publication year: 2011Source: Journal of Chromatography B, Available online 10 November 2011Ralf Koeber, Hans-Hermann Kluenemann, Reinhold Waimer, Anton Koestlbacher, Markus Wittmann, …A novel, simple, specific and sensitive high performance liq…

Publication year: 2011
Source: Journal of Chromatography B, Available online 10 November 2011

Ralf Koeber, Hans-Hermann Kluenemann, Reinhold Waimer, Anton Koestlbacher, Markus Wittmann, ...

A novel, simple, specific and sensitive high performance liquid chromatography (HPLC) assay for the detection and quantification of donepezil in serum of demented patients has been developed and validated. The analytical procedure involves an offline serum preextraction using solid phase extraction (SPE) cartridges (Oasis® HLB, Waters Co). The chromatographic analyses were performed on a Dionex HPLC system with a Phenomenex Luna Phenyl-Hexyl analytical column, and a mobile phase with the two components 0.02 mol/l phosphate buffer and acetonitrile. The flow rate was 0.4 ml/min. For the detection of donepezil three different UV wavelengths were used as an interference-control check. Interference tests between donepezil and 100 of the most commonly used concomitant medications allow quantification of donepezil under the polypharmaceutical conditions of the daily clinical routine. The retention time for donepezil was 12.1 min. The method was validated according to the guidelines of the Society of Toxicology and Forensic Chemistry (GTFCh): The calibration curve was linear over a concentration range from 5 to 160 ng/ml (n = 8/r>0.999). No endogenous compounds were found to interfere with the analyte, retention times for the comedication most often prescribed to demented patients are listed in the appendix. The method had an accuracy of >85%. Intra- and inter-assay coefficients of variation were <6% and <8%, respectively, at three different concentrations. The limit of quantification (LOQ) and the limit of detection (LOD) were found to be 6.1 and 1.7 ng/ml for donepezil. Application of the method to patient serum samples discovered that concentrations suggested as “therapeutic” in the literature may only be reached either by high, off-lable dosages or by utilization of inhibitory metabolic effects of the comedication.

Highlights

► The described procedure was the first assay to implement therapeutic drug monitoring of donepezil by HPLC/UV in medical routine analysis. The method reported here is simple, specific and sensitive. The UV absorption spectrum of donepezil showed maximal extinction at 210 nm, and three smaller extinction maxima at wavelengths of 235 nm, 268 nm and 315 nm. 210 nm was used as detection wavelength, two further wavelengths at 235 and 268 nm were used to double check for peak impurities. Interference tests between donepezil and 100 of the most commonly used concomitant medications allow quantification of donepezil under the polypharmaceutical conditions of the daily clinical routine. ► For economical reasons this assay has the capacity to be expanded to the qualitative and quantitative analysis of further drugs and their active metabolites relevant in the polypharmacy of demented patients, in particular other psychotropic drugs. ► Case histories of the AGATE pharmacovigilance program demonstrate that most of the dementia patients with donepezil treatment are underdosed. In “normal” cases off-label doses up to 40 mg/d donepezil will be necessary to reach the therapeutic concentrations suggested by Rogers et al., 1996. Such an approach can be supported by TDM as suggested in this paper.


Simple and rapid determination of thiol compounds by HPLC and fluorescence detection with 1,3,5,7-tetramethyl-8-phenyl- (2-maleimide) difluoroboradiaza-s-indacene

Publication year: 2011Source: Journal of Chromatography B, Available online 7 November 2011Xiao-Feng Guo, Pei-Xuan Zhao, Hong Wang, Hua-shan ZhangA rapid and simple background-free high-performance liquid chromatographic (HPLC) approach has been de…

Publication year: 2011
Source: Journal of Chromatography B, Available online 7 November 2011

Xiao-Feng Guo, Pei-Xuan Zhao, Hong Wang, Hua-shan Zhang

A rapid and simple background-free high-performance liquid chromatographic (HPLC) approach has been developed for simultaneously determining free thiol compounds including coenzyme A (CoA), cysteine (Cys), glutathione (GSH) and N-acetyl-cysteine (NAC) in biological samples by using 1,3,5,7-tetramethyl-8-phenyl-(2-maleimide) difluoroboradiaza-s-indacene (TMPAB-o-M) as fluorogenic reagent. After derivatization under physiological conditions within 6 min, baseline separation was finished in just 6 min using isocratic elution with reversed-phase HPLC and fluorescence detection. Excellent linearity was observed for all analytes over their concentration ranges of 1-500 nM and detection limits ranging 0.13 nM for CoA to 0.25 nM for Cys (S/N = 3) were achieved. The utility of the proposed method has been validated by measuring thiol compounds mentioned above in tissue, fluid and cell samples. The results indicated that this approach was well suited for high-throughput quantitative determination of thiols and study of the physiological role of them.

Highlights

► We develop an HPLC-fluorescence method for analysis of thiol compounds. ► The derivatization of coenzyme A, Cys, GSH and N-acetyl-cysteine need only 6 min. ► Baseline separation is achieved in 6 min using isocratic elution. ► The chromatograms are background-free. ► The detection limits are from 0.13 nM for coenzyme A to 0.25 nM for Cys (S/N = 3).


Simultaneous determination of codeine, ephedrine, guaiphenesin and chlorpheniramine in beagle dog plasma using high performance liquid chromatography coupled with tandem mass spectrometric detection: application to a bioequivalence study

Publication year: 2011Source: Journal of Chromatography B, Available online 7 November 2011Ziyan Hu, Qiaogen Zou, Jixin Tian, Lili Sun, Zunjian ZhangA rapid and sensitive method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) fo…

Publication year: 2011
Source: Journal of Chromatography B, Available online 7 November 2011

Ziyan Hu, Qiaogen Zou, Jixin Tian, Lili Sun, Zunjian Zhang

A rapid and sensitive method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous determination of codeine, ephedrine, guaiphenesin and chlorpheniramine in beagle dog plasma has been developed and validated. Following liquid-liquid extraction, the analytes were separated on a reversed-phase C18column (150mm × 2.0 mm, 3 μm) using formic acid:10 mM ammonium acetate: methanol (0.2:62:38, v/v/v) as mobile phase at a flow rate of 0.2 mL/min and analyzed by a triple-quadrupole mass spectrometer in the selected reaction monitoring (SRM) mode. The method was linear for all analytes over the following concentration (ng/mL) ranges: codeine 0.08∼16; ephedrine 0.8∼160; guaiphenesin 80∼16000; chlorpheniramine 0.2∼40. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. It is the first time that the validated HPLC–MS/MS method was successfully applied to a bioequivalence study in 6 healthy beagle dogs.

Highlights

► This is the first study to describe a sensitive and precise LC-MS/MS assay for codeine, ephedrine, guaiphenesin and chlorpheniramine. ► We utilized this method to measure concentration of codeine, ephedrine, guaiphenesin and chlorpheniramine in beagle dog plasma. ► This method supports research of codeine, ephedrine, guaiphenesin and chlorpheniramine and its pharmacokinetics in beagle dog plasma.


Screening for in vitro metabolites ofAbelmoschus manihotextract in intestinal bacteria by ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry

Publication year: 2011Source: Journal of Chromatography B, Available online 7 November 2011Caifu Xue, Shu Jiang, Jianming Guo, Dawei Qian, Jin-ao Duan, …Abelmoschus manihothas drawn much attention recently due to its potential beneficial health …

Publication year: 2011
Source: Journal of Chromatography B, Available online 7 November 2011

Caifu Xue, Shu Jiang, Jianming Guo, Dawei Qian, Jin-ao Duan, ...

Abelmoschus manihothas drawn much attention recently due to its potential beneficial health effects after oral administration. However, the metabolic fate ofAbelmoschus manihotin intestinal flora is not well understood. In this paper, we describe a strategy using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF MS) with automated data analysis software (MetaboLynx™) for fast analysis of the metabolic profile of flavonoids fromAbelmoschus manihotin intestinal flora. The human and rat incubated samples collected 72 h in the anaerobic incubator were analyzed by UPLC-Q-TOF MS within 10 min. A total of fourteen metabolites were identified in human and rat incubated solution compared with blank samples. The results indicated that hydrolysis, hydroxylation and acetylation were the major metabolic pathways of flavonoids inAbelmoschus manihotextract invitro. MSwas used for simultaneous acquisition of precursor ion information and fragment ion data at high and low collision energy in one analytical run, which facilitated the fast structural characterization of metabolites.This work demonstrated the potential of the UPLC-Q-TOF MS approach using Metabolynx for fast and automated identification of metabolites of natural product in intestinal flora.

Highlights

► Identification of the metabolites of flavonoids fromAbelmoschus manihotin intestinal flora. ► using UPLC-Q-TOF MS with automated data analysis software MetaboLynx™. ► A total of fourteen metabolites were identified in incubated solution. ► The results indicated that hydrolysis, hydroxylation and acetylation were the major metabolic pathways of flavonoids inAbelmoschus manihotextract.


Species discrimination of Radix Bupleuri through the simultaneous determination of ten saikosaponins by high performance liquid chromatography with evaporative light scattering detection and electrospray ionization mass spectrometry

Publication year: 2011Source: Journal of Chromatography B, Available online 4 November 2011Jaehyun Lee, Dong-Hyug Yang, Joon Hyuk Suh, Unyong Kim, Han Young Eom, …A simple, rapid and robust high performance liquid chromatography-evaporative ligh…

Publication year: 2011
Source: Journal of Chromatography B, Available online 4 November 2011

Jaehyun Lee, Dong-Hyug Yang, Joon Hyuk Suh, Unyong Kim, Han Young Eom, ...

A simple, rapid and robust high performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD) method was established for the species discrimination and quality evaluation of Radix Bupleuri through the simultaneous determination of ten saikosaponins, namely saikosaponin-a, -b1, -b2, -b3, -b4, -c, -d, -g, -h, and -i. These compounds were chromatographed on an AscentisExpress C18 column with a gradient elution of acetonitrile and water containing 0.1% acetic acid at a flow rate of 1.0 mL/min. Saikosaponins were monitored by ELSD, which was operated at a 50 °C drift tube temperature and 3.0 bar nebulizer gas (N2) pressure. The developed method was validated with respect to linearity, intra- and inter-day accuracy and precision, limit of quantification (LOQ), recovery, robustness and stability, thereby showing good precision and accuracy, with intra- and inter-assay coefficients of variation less than 15% at all concentrations. Furthermore, a high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS) method was developed to certify the existence of ten saikosaponins, as well as to confirm the reliability of ELSD. The extraction conditions of saikosaponins from Radix Bupleuri were also optimized by investigating the effect of extraction methods (sonication, reflux and maceration) and various solvents on the extraction efficiencies for saikosaponins. Sonication with 70% methanol for 40 min was found to be simple and effective for extraction of major saikosaponins. This analytical method was applied to determine saikosaponin profiles in 20 real samples consisting of fourBupleurumspecies, namelyB. falcatum, B. chinense,B. sibiricumand the poisonousB. longiradiatum. It was found that three major saikosaponin-a, -c and -d were the major constituents inB. falcatum, B. chinense, andB. longiradiatum, while one major saikosaponin (saikosaponins-c) was not identified fromB. sibiricum. In addition, no saikosaponin-b3was detected inB. longiradiatumsamples, indicating that the toxicB. longiradiatummay be tentatively distinguished from officially listedBupleurumspecies (B. falcatumandB. chinense) based on their saikosaponin profiles. Overall the simultaneous determination of ten saikosaponins in Radix Bupleuri was shown to be a promising tool to adopt for the discrimination and quality control of closely relatedBupleurumspecies.

Highlights

► We develop a HPLC-ELSD method for species discrimination of Radix Bupleuri. ► We analyze the ten saikosaponins simultaneously by HPLC-ELSD and LC-MS. ► The extraction condition of saikosaponins from Radix Bupleuri were optimized. ► The developed method was fully validated. ► This method was applied to determine saikosaponins in 20 Bupleurum species samples.


Biomimetic affinity purification ofCandida antarcticalipase B

Publication year: 2011Source: Journal of Chromatography B, Available online 4 November 2011Hongyan Yao, Tian Zhang, Hongwei Xue, Kexuan Tang, Rongxiu LiCandida antarcticalipase B (CalB) is one of the most widely used biocatalysts in organic synthe…

Publication year: 2011
Source: Journal of Chromatography B, Available online 4 November 2011

Hongyan Yao, Tian Zhang, Hongwei Xue, Kexuan Tang, Rongxiu Li

Candida antarcticalipase B (CalB) is one of the most widely used biocatalysts in organic synthesis. The traditional method for purification of CalB is a multi-step, high cost and low recovery procedure. Biomimetic affinity purification had high efficiency purification. We selected 298 ligand columns from a 700-member library of synthetic ligands to screenPichia pastorisprotein extract. Of the 298, three columns (named as A9-14, A9-10, and A11-33) had one-step purification effect, and A9-14 of these affinity ligands, had both high purification and recovery. The one-step recovery of CalB reached 73% and the purification reached 91% upon purification. The active groups of A9-14 were cyclohexylamine and propenylamine. Furthermore, both A9-14 and A9-10 had the same R1 active group of cyclohexylamine which might act the main binding role for CalB. The synthetic ligand A9-14 had a binding capacity of 0.4 mg/mL and had no negative effects on its hydrolytic activity. Unlike a natural affinity ligand, this synthetic ligand is highly stable to resist 1 M NaOH, and thus has great potential for industrial scale production of CalB.

Highlights

► Biomimetic affinity purification ofCandida antarcticalipase B ► One-step purification effect of ligand columns A9-14 ► Active groups of A9-14 were cyclohexylamine and propenylamine


Multiplex quantification of lamprey specific bile acid derivatives in environmental water using UHPLC-MS/MS

Publication year: 2011Source: Journal of Chromatography B, Available online 4 November 2011Ke Li, Huiyong Wang, Cory O. Brant, Sangchun Ahn, Weiming LiLarval and adult sea lampreys (Petromyzon marinus) release bile salts and acids into the surroun…

Publication year: 2011
Source: Journal of Chromatography B, Available online 4 November 2011

Ke Li, Huiyong Wang, Cory O. Brant, Sangchun Ahn, Weiming Li

Larval and adult sea lampreys (Petromyzon marinus) release bile salts and acids into the surrounding aquatic environment. Some of these bile salts and acids, such as petromyzonol sulfate (PZS), 3-keto petromyzonol sulfate (3k PZS), petromyzonamine disulfate (PADS), petromyzosterol disulfate (PSDS), and 3-keto allocholic acid (3k ACA), may function as pheromones. To examine the release and distribution patterns of these metabolites, which this study has termed bile acid derivatives, we developed a novel UHPLC–MS/MS method that was characterized by simple sample preparation, baseline separation, and short analysis time for all studied compounds. These five analytes were separated in 7 minutes using a reversed-phase C18 column containing 1.7 μm particles and a gradient elution at pH 8.9. Once separated, the analytes were subjected to electrospray ionization-mass spectrometry (negative ion mode) and collision-induced dissociation tandem mass spectrometry (CID-MS/MS) using the multiple reaction monitoring (MRM) mode. Deuterated 3kPZS ([H5]3kPZS) was added as the internal standard (IS) to the sample prior to solid phase extraction (SPE). Among the three types of SPE sorbent tested, mixed-mode cation-exchange and reversed-phase sorbent for bases (MAX) and acids (MCX), and reversed-phase C18 sorbent (Sep-pak), the best recoveries (84.2 ∼ 97.6%) were obtained with MCX cartridges. The calibration curves of all five analytes were linear between 0.15 and 1200 ng/mL, with R≥ 0.9997. This method had a precision of relative standard deviation (RSD) ≤ 9.9% and an accuracy of deviation (DEV) ≥ 92.5%. The developed method was successfully used to quantify bile acid derivatives found in streams where lampreys spawn (SD < 1.4) and water conditioned with male sea lampreys (SD < 4.8). Utilizing this method provides a routine analysis of lamprey bile acid derivatives and may prove useful for sea lamprey population estimates in future studies and applications.

Highlights

► Simultaneous measurement of bile acid derivatives in environmental water and animal conditioned samples. ► Using of MCX cartridge in sample process resulted in excellent recoveries. ► UHPLC/MS/MS accomplished high-resolution separation and provided lower LODs. ► The developed method is an appealing methodology for routine analysis.


A rapid and sensitive method for the analysis of brain monoamine neurotransmitters using ultra-fast liquid chromatography coupled to electrochemical detection

Publication year: 2011Source: Journal of Chromatography B, Available online 3 November 2011Sandrine Parrot, Pierre-Charles Neuzeret, Luc DenoroyElectrochemical detection is often used to detect catecholamines and indolamines in brain samples that ha…

Publication year: 2011
Source: Journal of Chromatography B, Available online 3 November 2011

Sandrine Parrot, Pierre-Charles Neuzeret, Luc Denoroy

Electrochemical detection is often used to detect catecholamines and indolamines in brain samples that have been separated by conventional reverse-phase high performance liquid chromatography (HPLC). The present paper presents the transfer of an existing chromatographic method for the determination of monoamines in brain tissues using 5 μm granulometry HPLC columns to columns with a particle diameter less than 3 μm. Several parameters (repeatability, linearity, accuracy, limit of detection, and stability of samples) for this new ultrafast high performance liquid chromatography (UHPLC) method were examined after optimization of the analytical conditions. The separation of seven compounds, noradrenaline, dopamine and three of its metabolites, dihydroxyphenylacetic acid, homovanillic acid, and 3-methoxytyramine, and serotonin and its metabolite, 5-hydroxyindole-3-acetic acid was analyzed using this UHPLC-electrochemical detection method. The final method, which was applied to brain tissue extracts from mice, rats, and cats, decreased analysis time by a factor of 4 compared to HPLC, while guaranteeing good analytical performance.

Highlights

► Catecholamines and indolamines as the most studied monoamines in neuroscience. ► Time of routine analyses by conventional HPLC and electrochemical detection is too long ► Use of sub-2 μm particles increases the throughput of analysis using ultra-fast HPLC ► Brain monoamines can be determined in various tissue samples by this way ► Potential applications of ultra-fast HPLC in brain dialysis.


Determination of benzodiazepines in ante-mortem and post-mortem whole blood by solid-supported liquid-liquid extraction and UPLC-MS/MS

Publication year: 2011Source: Journal of Chromatography B, Available online 3 November 2011E.N. Sauve, M. Langødegård, D. Ekeberg, Å.M.L. ØiestadA solid-supported liquid-liquid extraction ultra performance liquid chromatography tandem mass spec…

Publication year: 2011
Source: Journal of Chromatography B, Available online 3 November 2011

E.N. Sauve, M. Langødegård, D. Ekeberg, Å.M.L. Øiestad

A solid-supported liquid-liquid extraction ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the determination of benzodiazepines commonly found in Norway, for use in cases with suspected driving impairment and autopsy cases by analysis of human whole blood samples. The following compounds were included: alprazolam, bromazepam, clonazepam, diazepam, flunitrazepam, lorazepam, midazolam, nitrazepam, nordiazepam (metabolite of diazepam), oxazepam and phenazepam.Aliquots of 500 μL whole blood were added 500 μL of borate buffer pH 11 and extracted by solid-supported liquid-liquid extraction on ChemElut® columns using three times 2.5 mL of methyltert-butyl ether. Deuterated analogues were used as internal standards (IS) for all analytes, except for midazolam, phenazepam and bromazepam which had no commercially available deuterated analogues at the time the method was developed, and therefore used diazepam-d5, flunitrazepam-d7and nitrazepam-d5,respectively.The analytes were separated using UPLC with a 2.1 × 100 mm BEH C18-column, 1.7 μm particle size, and quantified by MS/MS using multiple reaction monitoring (MRM) in positive mode. Two transitions were used for the analytes and one transition for the IS. The run time of the method was 8 minutes including equilibration time.The concentrations of the benzodiazepines in the method span a broad range varying from the lowest concentration of 0.005 μM for flunitrazepam to the highest of 20 μM for oxazepam. The calibration curves of extracted whole blood standards were fitted by second-order calibration curves weighted 1/x, with Rvalues ranging from 0.9981 to 0.9998. The intermediate precision had a CV (%) ranging between 2 and 19%. Recoveries of the analytes were from 71 to 96%. The LLOQs for the analytes varied from 0.0006 to 0.075 μM and the LODs from 0.005 to 3.0 nM. Matrix effects were studied by post extraction addition and found to be between 95–104% when calculated against an internal standard. A comparison with two other LC-MS methods was performed during method validation. Good correlation was seen for all analytes. The method has been running on a routine basis for several years, and has proven to be very robust and reliable with good results for external quality samples. The method also meets the requirements of the legislative limits for driving under the influence of non-alcohol drugs to be introduced in the Norwegian legislative system from 2012.

Purification of lipase produced by a new source ofBacillusin submerged fermentation using an aqueous two-phase system

Publication year: 2011Source: Journal of Chromatography B, Available online 3 November 2011José Murillo P. Barbosa, Ranyere L. Souza, Alini T. Fricks, Gisella Maria Zanin, Cleide Mara F. Soares, …This work discusses the application of an aqueou…

Publication year: 2011
Source: Journal of Chromatography B, Available online 3 November 2011

José Murillo P. Barbosa, Ranyere L. Souza, Alini T. Fricks, Gisella Maria Zanin, Cleide Mara F. Soares, ...

This work discusses the application of an aqueous two-phase system for the purification of lipases produced byBacillussp. ITP-001 using polyethylene glycol (PEG) and potassium phosphate. In the first step, the protein content was precipitated with ammonium sulphate (80% saturation). The enzyme remained in the aqueous solution and was dialyzed against ultra-pure water for 18 h and used to prepare an aqueous two-phase system (PEG/potassium phosphate). The use of different molecular weights of PEG to purify the lipase was investigated; the best purification factor (PF) was obtained using PEG 20,000 g/mol, however PEG 8,000 was used in the next tests due to lower viscosity. The influence of PEG and potassium phosphate concentrations on the enzyme purification was then studied: the highest FP was obtained with 20% of PEG and 18% of potassium phosphate. NaCl was added to increase the hydrophobicity between the phases, and also increased the purification factor. The pH value and temperature affected the enzyme partitioning, with the best purifying conditions achieved at pH 6.0 and 4 °C. The molecular mass of the purified enzyme was determined to be approximately 54 kDa by SDS-PAGE. According to the results the best combination for purifying the enzyme is PEG 8,000 g/mol and potassium phosphate (20/18%) with 6% of NaCl at pH 6.0 and 4 °C (201.53 fold). The partitioning process of lipase is governed by the entropy contribution.

Highlights

► ATPS could be used to selectively purify lipase from the crude culture filtrates. ► The best purification factor was 201.53 fold. ► The enzyme partitioning is governed by entropy. ► Lipase byBacillussp. ITP-001 purified in PEG/potassium phosphate ATPS has 54 kDa.


High-performance liquid chromatographic determination of tiapride and itsphase Imetabolite in blood plasma using tandem UV photodiode-array and fluorescence detection

Publication year: 2011Source: Journal of Chromatography B, Available online 3 November 2011Milan Nobilis, Zuzana Vybíralová, Barbora Szotáková, Květa Sládková, Martin Kuneš, …New bioanalytical SPE-HPLC-PDA-FL method for the determination…

Publication year: 2011
Source: Journal of Chromatography B, Available online 3 November 2011

Milan Nobilis, Zuzana Vybíralová, Barbora Szotáková, Květa Sládková, Martin Kuneš, ...

New bioanalytical SPE-HPLC-PDA-FL method for the determination of the neuroleptic drug tiapride and itsN-desethyl metabolite was developed, validated and applied to xenobiochemical and pharmacokinetic studies in humans and animals.The sample preparation process involved solid-phase extraction of diluted plasma spiked with sulpiride (an internal standard) using SPE cartridges DSC-PH Supelco, USA. Chromatographic separation of the extracts was performed on a Discovery HS F5 250 × 4 mm (Supelco) column containing pentafluorophenylpropylsilyl silica gel. Mobile phase (acetonitrile–0.01 Mphosphate buffer pH = 3, flow rate 1 ml.min) in the gradient mode was employed in the HPLC analysis. Tandem UV photodiode-array → fluorescence detection was used for the determination of the analytes. Low concentrations of tiapride andN-desethyl tiapride were determined using a more selective fluorescence detector (λ(exc.)/λ(emis.) = 232 nm/334 nm), high concentrations (500 - 6000 pmol.ml) using a UV PDA detector at 212 nm with a linear response. Each HPLC run lasted 15 minutes. Lower limits of quantification (LLOQ) for tiapride (N-desethyl tiapride) were found to be 8.24 pmol.ml(10.11 pmol.ml). The recoveries of tiapride ranged from 89.3 - 94.3%, 81.7 - 86.8% for internal standard sulpiride and 90.9 - 91.8% forN-desethyl tiapride.

Highlights

► In the manuscript, the synthesis of the standard of principal tiapride metabolite (N-desethyl tiapride) including the MS-MS confirmation of its chemical structure was described. ► Standards of N-desethyl tiapride, sulpiride (I.S.) and tiapride were used in the development and validation of new bioanalytical SPE-HPLC-PDA-FL method for the determination of tiapride and its metabolite in biomatrices using data from both UV photodiode-array and fluorescence detectors in tandem. ► New SPE method increased recovery of the metabolite 9-times in comparison with formerly reported results.


Simplified absolute metabolite quantification by gas chromatography-isotope dilution mass spectrometry on the basis of commercially available source material

Publication year: 2011Source: Journal of Chromatography B, Available online 3 November 2011Oliver Vielhauer, Maksim Zakhartsev, Thomas Horn, Ralf Takors, Matthias ReussIn the field of metabolomics, GC-MS has rather established itself as a tool for…

Publication year: 2011
Source: Journal of Chromatography B, Available online 3 November 2011

Oliver Vielhauer, Maksim Zakhartsev, Thomas Horn, Ralf Takors, Matthias Reuss

In the field of metabolomics, GC-MS has rather established itself as a tool for semi-quantitative strategies like metabolic fingerprinting or metabolic profiling. Absolute quantification of intra- or extracellular metabolites is nowadays mostly accomplished by application of diverse LC-MS techniques. Only few groups have so far adopted GC-MS technology for this exceptionally challenging task. Besides numerous and deeply investigated problems related to sample generation, the pronounced matrix effects in biological samples have led to the almost mandatory application of isotope dilution mass spectrometry (IDMS) for the accurate determination of absolute metabolite concentrations. Nevertheless, access to stable isotope labeled internal standards (ILIS), which are in many cases commercially unavailable, is quite laborious and very expensive.Here we present an improved and simplified gas chromatography - isotope dilution mass spectrometry (GC-IDMS) protocol for theabsolutedetermination of intra- and extracellular metabolite levels. Commercially availableC-labeled algal cells were used as a convenient source for the preparation of internal standards. Advantages as well as limitations of the described method are discussed.

Highlights

► It was demonstrated that GC-IDMS is suitable for the accurateabsolutequantification of metabolites in microbial cultures. ► The described method exhibits certain attractive advantages over established LC-IDMS protocols. ► Certain metabolites were subjected toabsolutequantification by GC-IDMS for the first time. ► Examination of derivatization conditions and sample matrix effects resulted in distinct improvements as well as practical recommendations. ► First-time use of commercially available U-C-labeled algal cells as a convenient source for the generation of isotope labeled internal standards.


Quantification of bioactive sphingo- and glycerophospholipid lipid species by electrospray ionization tandem mass spectrometry in blood

Publication year: 2011Source: Journal of Chromatography B, Available online 3 November 2011Gerhard Liebisch, Max SchererBioactive glycerophospho- and sphingolipids species are involved in the regulation of numerous biological processes and implicated…

Publication year: 2011
Source: Journal of Chromatography B, Available online 3 November 2011

Gerhard Liebisch, Max Scherer

Bioactive glycerophospho- and sphingolipids species are involved in the regulation of numerous biological processes and implicated in the pathophysiology of various diseases. Here we review electrospray ionization tandem mass spectrometric (ESI-MS/MS) methods for the analysis of these bioactive lipid species in blood including lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC), bis(monoacylglycero)phosphate (BMP), ceramide (Cer), sphingosine-1-phosphate (S1P) and sphingosylphosphorylcholine (SPC). Beside direct tandem mass spectrometric and liquid chromatography coupled approaches, we present an overview of concentrations of these bioactive lipids in plasma. The analytical strategies are discussed together with aspects of sample preparation, quantification and sample stability.

Successful and cost-efficient replacement of immunoassays by tandem mass spectrometry for the quantification of immunosuppressants in the clinical laboratory

Publication year: 2011Source: Journal of Chromatography B, Available online 3 November 2011Pierre-Olivier Hétu, Robert Robitaille, Bernard VinetThe purpose of this paper is to describe the implantation of mass spectrometry in replacement of immunoa…

Publication year: 2011
Source: Journal of Chromatography B, Available online 3 November 2011

Pierre-Olivier Hétu, Robert Robitaille, Bernard Vinet

The purpose of this paper is to describe the implantation of mass spectrometry in replacement of immunoassays for the measurement of immunosuppressant drugs in the clinical setting, from scientific and financial perspectives. A straightforward, rapid, and economical method was developed for the simultaneous quantification of tacrolimus, sirolimus, and cyclosporine. Following a simple protein precipitation step, supernatants are injected on a small C18guard cartridge and gradient elution of the immunosuppressants is performed in a total chromatographic run time of 2.25 min. Sodium adducts of the compounds and internal standards are quantified by electrospray tandem mass spectrometry. The method shows inter-assay impression of less than 10-15% for all compounds with good extraction efficiency (89-104%) and minimal matrix effects, except for sirolimus where ion suppression is more pronounced. The method correlates well with chemiluminescent microparticle immunoassays (on the Abbott Architect analyzer), although the immunoassays results are significantly higher than those obtained by HPLC-MS/MS. The transition from immunoassays to mass spectrometry was well received by the laboratory staff, and significant reductions in reagent costs have been realized (> $250,000 CAD per year). With these savings, the purchase and installation of two complete HPLC-MS/MS systems was completely financed in less than three years.

Determination of Cediranib in Mouse Plasma and Brain Tissue Using High-Performance Liquid Chromatography-Mass Spectrometry

Publication year: 2011Source: Journal of Chromatography B, Available online 29 October 2011Tianli Wang, Rajneet K. Oberoi, William F. ElmquistA new high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) assay for c…

Publication year: 2011
Source: Journal of Chromatography B, Available online 29 October 2011

Tianli Wang, Rajneet K. Oberoi, William F. Elmquist

A new high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) assay for cediranib, a tyrosine kinase inhibitor for VEGFRs, was developed and validated, for the determination of plasma and brain levels of cediranib in small specimen volumes. Tyrphostin (AG1478) was used as internal standard. Mouse plasma and brain homogenate samples were prepared using liquid-liquid extraction. The assay was validated for a 2.5-2500 ng/mL concentration range for plasma, and for 1-2000 ng/mL range for brain homogenate. For these calibration ranges, within-assay variabilities were 1.1-14.3% for plasma and 1.5-9.4% for brain homogenate; between-assay variabilities were 2.4-9.2% for plasma, and 4.9-10.2% for brain homogenate. Overall accuracy ranged from 101.5 to 107.0% for plasma and 96.5 to 100.2% for brain homogenate, for all target concentrations. The developed assay has been successfully applied for a mouse brain distribution study in mice at an oral dose of 5 mg/kg.

Highlights

► This is the first study to describe a sensitive and precise LC-MS assay for cediranib. ► This assay allows small specimen volumes and adequate detectability to use in tissue distribution studies. ► We have employed the assay in a brain distribution study in the mouse, to examine different mechanisms that limit the CNS distribution of cediranib. ► The assay is generally applicable to a variety of pharmacokinetic and in vitro cell culture experiments


A LC-MS/MS METHODOLOGY TO DETERMINE FURALTADONE RESIDUES IN THE MACROALGAEULVA LACTUCA

Publication year: 2011Source: Journal of Chromatography B, Available online 29 October 2011Sara Leston, Margarida Nunes, Andreia Freitas, Jorge Barbosa, Fernando Ramos, …Presently, the rise of new contaminants in the environment has widened the …

Publication year: 2011
Source: Journal of Chromatography B, Available online 29 October 2011

Sara Leston, Margarida Nunes, Andreia Freitas, Jorge Barbosa, Fernando Ramos, ...

Presently, the rise of new contaminants in the environment has widened the scope of pharmaceutical analyses as to face the demanding new challenges. An increasing tendency for the interconnection and overlap of research fields, such as ecology and biochemistry, is intensifying the demand for new methodologies to be applied to the survey of drugs in unconventional matrices. Integrated in this group are macrophytes, such as the green macroalgaeUlva lactuca, which are under study as to ascertain their ability as indicators of contamination for many substances. Nonetheless, methodologies for extraction and determination of drugs in such matrices are scarce and new studies on the subject are pressing. A new methodology for the determination of the antibiotic furaltadone inU. lactucaby liquid chromatography-tandem mass spectrometric (LC-MS/MS) procedure was developed, optimized and validated following the guidelines of the EC Decision 2002/657. The calibration curves showed linearity above 0.99 (R). The relative standard deviations obtained for repeatability, expressed as CV, were between 15.3 and 20.5 and for reproducibility 25.3 and 28.2 whereas accuracy was in the interval of 88.9 to 95.5 (%). The limit of decision (CCα) and the detection capability (CCβ) were respectively 5.57 μg kgand 10.97 μg kg. The method was successfully applied to experimental samples.

Highlights

► New methodology to detect and quantify the nitrofuran antibiotic furaltadone in the green macroalgaeUlva lactuca► Method relies on LC-MS/MS techniques ► experimental assay with exposure ofU lactucato hypothetical prophylactic and therapeutic concentrations ► successfully applied to blank spiked and experimental samples