Improving the Sensitivity of Mass Spectrometry by Using a New Sheath Flow Electrospray Emitter Array at Subambient Pressures

Arrays of chemically etched emitters with individualized sheath gas capillaries were developed to enhance electrospray ionization (ESI) efficiency at subambient pressures. By incorporating the new emitter array in a subambient pressure ionization with nanoelectrospray (SPIN) source, both ionization efficiency and ion transmission efficiency were significantly increased, providing enhanced sensitivity in mass spectrometric analyses. The SPIN source eliminates the major ion losses of conventional ESI-mass spectrometry (MS) interfaces by placing the emitter in the first reduced pressure region of the instrument. The new ESI emitter array design developed in this study allows individualized sheath gas around each emitter in the array making it possible to generate an array of uniform and stable electrosprays in the subambient pressure (10 to 30 Torr) environment for the first time. The utility of the new emitter arrays was demonstrated by coupling the emitter array/SPIN source with a time of flight (TOF) mass spectrometer. The instrument sensitivity was compared under different ESI source and interface configurations including a standard atmospheric pressure single ESI emitter/heated capillary, single emitter/SPIN and multi-emitter/SPIN configurations using an equimolar solution of nine peptides. The highest instrument sensitivity was observed using the multi-emitter/SPIN configuration in which the sensitivity increased with the number of emitters in the array. Over an order of magnitude MS sensitivity improvement was achieved using multi-emitter/SPIN compared with using the standard atmospheric pressure single ESI emitter/heated capillary interface. Graphical Abstract

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Determination of tandospirone in human plasma by a liquid chromatography–tandem mass spectrometry method

A rapid, sensitive, and selective liquid chromatography–tandem mass spectrometry method for the detection of tandospirone in human plasma is described. It was employed in a pharmacokinetic study. The analyte and internal standard diphenhydramine were extracted from plasma using liquid–liquid extraction, then separated on a Zorbax XDB C₁₈ column using a mobile phase of methanol–water–formic acid (80:20:0.5, v/v/v). The detection was performed with a tandem mass spectrometer equipped with an electrospray ionization source. Linearity was established in the concentration range of 10.0-5,000 pg/ml. The lower limit of quantification was 10.0 pg/ml. The intraday and interday relative standard deviation across three validation runs over the entire concentration range was less than 13%. Accuracy determined at three concentrations (25.0, 200, and 4,000 pg/ml for tandospirone) ranged from 94.4 to 102.1%. Each plasma sample was chromatographed within 3.4 min. The method proved to be highly selective and suitable for bioequivalence evaluation of different formulations containing tandospirone and clinical pharmacokinetic investigation of tandospirone.     

Detection of positive and negative ions from a flowing atmospheric pressure afterglow using a mattauch-herzog mass spectrograph equipped with a faraday-strip array detector

An ambient desorption/ionization (ADI) source, known as the flowing atmospheric pressure afterglow (FAPA), has been coupled to a Mattauch-Herzog mass spectrograph (MHMS) equipped with a focal plane camera (FPC) array detector. The FAPA ionization source enables direct mass spectral analysis of solids, liquids, and gases through either positive or negative ionization modes. In either case, spectra are generally simple with dominant peaks being the molecular ions or protonated molecular ions. Use of the FAPA source with the MHMS allows the FPC detector to be characterized for the determination of molecular species, whereas previously only atomic mass spectrometry (MS) has been demonstrated. Furthermore, the FPC is shown to be sensitive to negative ions without the need to change any detector parameters. The analysis of solid, liquid, and gaseous samples through positive and negative ionization is demonstrated with detection limits (1–25 fmol/s, ∼0. 3–10 pg of analyte per mL of helium) surpassing those obtained with the FAPA source coupled to a time-of-flight mass analyzer.     

Phytoestrogen biomonitoring: an extractionless LC-MS/MS method for measuring urinary isoflavones and lignans by use of atmospheric pressure photoionization (APPI)

We present here a high-performance liquid chromatography−tandem mass spectrometry (LC-MS/MS) method for quantifying phytoestrogenic isoflavones (daidzein, equol, genistein, and O-desmethylangolensin) and lignans (enterodiol and enterolactone) in urine without the use of extraction or the preconcentration techniques inherent in existing methods. The development of this concept was made possible by use of atmospheric pressure photoionization (APPI); an ionization technique that we found to improve analyte sensitivity relative to electrospray ionization and atmospheric pressure chemical ionization for this particular group of compounds. The analytical performance of this method was equal to or exceeded that of comparable methods. Between-run coefficients of variation (CVs) across three quality control (QC) pool levels analyzed in duplicate over 20 days were 3.1–5.8% CV; within-run CVs were 2.3–6.0%. Accuracy, as determined by average spike recovery in QC pools, was generally within ±10% of being quantitative (100%). Relative limits of detection were 0.04–0.4 ng/mL urine, with absolute detection limits as low as 0.1 pg. This method was applied to the analysis of >2,500 urine specimens for the 2005–2006 Centers for Disease Control and Prevention’s National Health and Nutrition Examination Survey (NHANES). The method was capable of quantifying these compounds in 95–100% of study samples. This work is the first ever report of using APPI for the LC-MS/MS determination of these compounds in urine. It is also the first method of its kind to do so without any need for analyte extraction or preconcentration prior to analysis.     

Development of a UPLC-ESI-MS/MS method for the determination of larotaxel in beagle dog plasma: application to the pharmacokinetic study

A UPLC-ESI-MS/MS method has been developed and validated for the determination of larotaxel in beagle dog plasma. After addition of the internal standard, plasma samples were extracted by liquid–liquid extraction with methyl tert-butyl ether and separated on a 50 × 2.1 mm ACQUITY 1.7 μm C₁₈ column (Waters, USA), with acetonitrile and 5 mM ammonium acetate as mobile phase, within a runtime of 3.0 min. The analytes were detected without interference in Multiple Reaction Monitoring mode with positive electrospray ionization. The linear range was 2.5–5,000 ng/mL. The intra-day and inter-day precisions (relative standard deviation, RSD, %) were within 9.3% and 10.2%, respectively, and the accuracy (relative error, RE, %) was less than 11.5%. The validated method was successfully applied to a pharmacokinetic study of larotaxel in beagle dogs after intravenous administration of larotaxel-loaded lipid microsphere with different doses of 0.4, 0.8, and 1.6 mg/kg. The area under the concentration–time curve and the peak concentration of larotaxel seemed to increase with increasing dose proportionally, suggesting linear pharmacokinetics.     

Liquid sampling–atmospheric pressure glow discharge (LS-APGD) ionization source for elemental mass spectrometry: preliminary parametric evaluation and figures of merit

A new, low-power ionization source for the elemental analysis of aqueous solutions has recently been described. The liquid sampling–atmospheric pressure glow discharge (LS-APGD) source operates at relatively low currents (<20 mA) and solution flow rates (<50 μL min⁻¹), yielding a relatively simple alternative for atomic mass spectrometry applications. The LS-APGD has been interfaced to what is otherwise an organic, LC-MS mass analyzer, the Thermo Scientific Exactive Orbitrap without any modifications, other than removing the electrospray ionization source supplied with that instrument. A glow discharge is initiated between the surface of the test solution exiting a glass capillary and a metallic counter electrode mounted at a 90° angle and separated by a distance of ~5 mm. As with any plasma-based ionization source, there are key discharge operation and ion sampling parameters that affect the intensity and composition of the derived mass spectra, including signal-to-background ratios. We describe here a preliminary parametric evaluation of the roles of discharge current, solution flow rate, argon sheath gas flow rate, and ion sampling distance as they apply on this mass analyzer system. A cursive evaluation of potential matrix effects due to the presence of easily ionized elements indicate that sodium concentrations of up to 50 μg mL⁻¹ generally cause suppressions of less than 50%, dependant upon the analyte species. Based on the results of this series of studies, preliminary limits of detection (LOD) have been established through the generation of calibration functions. While solution-based concentration LOD levels of 0.02–2 μg mL⁻¹ are not impressive on the surface, the fact that they are determined via discrete 5 μL injections leads to mass-based detection limits at picogram to single-nanogram levels. The overhead costs associated with source operation (10 W d.c. power, solution flow rates of <50 μL min⁻¹, and gas flow rates <10 mL min⁻¹) are very attractive. While further optimization in the source design is suggested here, it is believed that the LS-APGD ion source may present a practical alternative to inductively coupled plasma sources typically employed in elemental mass spectrometry.     

Uniformity of ionization response of structurally diverse analytes using a chip-based nanoelectrospray ionization source

The major drawback of liquid chromatography/mass spectrometry (LC/MS) for the analysis of mixtures is the non-quantitative nature of these studies. The ionization efficiency of the various components in the mixture (e.g., a compound and its metabolites) can vary greatly and, therefore, relative intensities of signals cannot be related to relative abundance. A chip-based nanoelectrospray ionization source was used to compare the ionization efficiencies of compounds with different physical-chemical characteristics. The data indicate that the ionization efficiencies vary much less with the chip-based device than by LC/MS. This was ascribed to the generation of a much higher electric field around the nozzles, which supplies a large excess of protons to the small droplets and reduces/eliminates the differences in the ionization efficiency for the analytes.     

A screening method for the simultaneous detection of glucocorticoids,diuretics, stimulants,anti-oestrogens,beta-adrenergic drugs and anabolic steroids in human urine by LC-ESI-MS/MS

This paper presents a general screening method, based on liquid chromatography/mass spectrometry (LC/MS), for the simultaneous detection in human urine of 72 xenobiotics (21 diuretics, 16 synthetic glucocorticoids, 17 beta-adrenergic drugs, 10 stimulants, 5 anti-oestrogens and 3 anabolic steroids), excreted free or as glucuro-conjugates in urine. Although the method has been specifically designed and evaluated in view of its potential application to anti-doping analyses, it can also be effective in other areas of analytical toxicology. Sample preparation was based on two liquid/liquid separation steps (performed at alkaline and at acid pH, respectively) of hydrolyzed human urine, and then an assay by LC/MS-MS in positive and negative ionization mode using an electrospray ionization source (ESI) and multiple reaction monitoring (MRM) as the acquisition mode. The overall time needed for an LC run was less than 15 minutes. All compounds showed good reproducibility in terms of both the retention times (CV%<1) and the relative abundances of the diagnostic transitions (CV%<10). The limits of detection (LOD) were in the range of 1–50 ng/mL for glucocorticoids, anti-oestrogens and steroids, and 50–500 ng/mL for diuretics, beta-adrenergic drugs and stimulants, thus satisfying the minimum required performance limits (MRPL) set by the World Anti-Doping Agency (WADA) for the accredited anti-doping laboratories.     

Spectroscopic Characterization of Miniaturized Atmospheric-Pressure dc Glow Discharge Generated in Contact with Flowing Small Size Liquid Cathode

The miniaturized atmospheric pressure glow discharge (APGD) generated between a solid electrode and a flowing small size liquid cathode (dimension 2 mm) was investigated here using optical emission spectroscopy. The discharge was studied in an open air atmosphere, and the spectral characteristics of the plasma source was examined. Analysed APGD was operated at a discharge voltage of 1,100–1,700 V, a discharge current of 20 mA and gaps between a solid anode and a liquid cathode in the range from 0.5 to 3.5 mm. The emission intensities of the main species were measured as a function of various experimental conditions, including the solution flow rate, the gap between the electrodes, and the concentration of hydrochloric acid. The excitation temperature, the vibrational temperatures calculated from N₂, OH, and NO bands, and the rotational temperatures determined from band of OH, N₂ and NO, were found to be dependent on these experimental parameters. The electron number density was determined from the Stark broadening of H_β line. Additionally, the ionization temperature and degree were calculated using the Saha–Boltzmann equation, with the ion to atom ratio for magnesium (MgII/MgI). The results demonstrated that T _exc(H), T _vib(N₂), T _vib(OH), T _vib(NO) and T _rot(OH) were well comparable (~3,800–4,200 K) for selected plasma generation conditions (gap ≥2.5 mm, HCl concentration ≥0.1 mol L⁻¹), while the rotational temperatures determined from band of N₂ (~1,700–2,100 K) and band of NO (~3,000 K) were considerably lower. The electron number density was evaluated to be (3.4–6.8) × 10²⁰ m⁻³ and the ionization temperature varied, throughout in the 4,900–5,200 K range.     

Infrared Laser Ablation Sample Transfer for MALDI and Electrospray

We have used an infrared laser to ablate materials under ambient conditions that were captured in solvent droplets. The droplets were either deposited on a MALDI target for off-line analysis by MALDI time-of-flight mass spectrometry or flow-injected into a nanoelectrospray source of an ion trap mass spectrometer. An infrared optical parametric oscillator (OPO) laser system at 2.94 μm wavelength and approximately 1 mJ pulse energy was focused onto samples for ablation at atmospheric pressure. The ablated material was captured in a solvent droplet 1–2 mm in diameter that was suspended from a silica capillary a few millimeters above the sample target. Once the sample was transferred to the droplet by ablation, the droplet was deposited on a MALDI target. A saturated matrix solution was added to the deposited sample, or in some cases, the suspended capture droplet contained the matrix. Peptide and protein standards were used to assess the effects of the number of IR laser ablation shots, sample to droplet distance, capture droplet size, droplet solvent, and laser pulse energy. Droplet collected samples were also injected into a nanoelectrospray source of an ion trap mass spectrometer with a 500 nL injection loop. It is estimated that pmol quantities of material were transferred to the droplet with an efficiency of approximately 1%. The direct analysis of biological fluids for off-line MALDI and electrospray was demonstrated with blood, milk, and egg. The implications of this IR ablation sample transfer approach for ambient imaging are discussed.     

Sensitivity “Hot Spots” in the Direct Analysis in Real Time Mass Spectrometry of Nerve Agent Simulants

Presented here are findings describing the spatial-dependence of sensitivity and ion suppression effects observed with direct analysis in real time (DART). Continuous liquid infusion of dimethyl methyl phosphonate (DMMP) revealed that ion yield “hot spots” did not always correspond with the highest temperature regions within the ionization space. For instance, at lower concentrations (50 and 100 μM), the highest sensitivities were in the middle of the ionization region at 200 °C where there was a shorter ion transport distance, and the heat available to thermally desorb neutrals was moderate. Conversely, at higher DMMP concentrations (500 μM), the highest ion yield was directly in front of the DART source at 200 °C where it was exposed to the highest temperature for thermal desorption. In matching experiments, differential analyte volatility was observed to play a smaller role in relative ion suppression than differences in proton affinity and the relative sampling positions of analytes. At equimolar concentrations sampled at the same position, suppression was as high as 26× between isoquinoline (proton affinity 952 kJ mol^–1, boiling point 242 °C) and p-anisidine (proton affinity 900 kJ mol^–1, boiling point 243 °C). This effect was exacerbated when sampling positions of the two analytes differed, reaching levels of relative suppression as high as 4543.0× ± 1406.0. To mitigate this level of relative ion suppression, sampling positions and molar ratios of the analytes were modified to create conditions in which ion suppression was negligible.     

HPLC-CHIP coupled to a triple quadrupole mass spectrometer for carbonic anhydrase II quantification in human serum

A method for carbonic anhydrase II (CA II) absolute quantification in human serum is presented. This method is based on high-performance liquid chromatography (HPLC)-Chip microfluidic device incorporating a nanoelectrospray source interfaced to a triple quadrupole mass spectrometer. The fraction containing CA II was isolated by preparative reversed-phase HPLC, and peptides obtained from the tryptic digest of the protein mixture were separated by the HPLC-Chip system. The multiple-reaction monitoring acquisition mode of a selected suitable CA II peptide and peptide internal standard allowed the selective and sensitive determination of a CA II. Absolute recovery of the method was 52 ± 12%, while analytical recovery was 81 ± 10%. For the eight samples analyzed, the matrix effect was found to be only −14 ± 6%. A comparison among three regression lines type which were obtained by external calibration, matrix-matched calibration, and standard addition method, respectively, demonstrated that the first one is adequate in obtaining good accuracy and precision. Method quantification limit for CA II in serum was estimated to be 2 fmol/mL. CA II mean concentration in sera from eight healthy subjects was found to be 56 pmol/mL (relative standard deviation 24%).     

A Potential Commercial Source of Fucoxanthin Extracted from the Microalga <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis>

Fucoxanthin, one of the main marine carotenoids, is abundant in macro- and microalgae. Here, fucoxanthin was isolated and structurally identified as the major carotenoid in the diatom Phaeodactylum tricornutum through chromatographic and spectroscopic methods, such as liquid chromatography–positive-ion atmospheric pressure chemical ionization/mass spectroscopy and nuclear magnetic resonance. This pigment was quantified by reverse-phase high-performance liquid chromatography, and a number of extraction procedures were assessed to investigate the effect of solvent type, extraction time, temperature, and extraction method (maceration, ultrasound-assisted extraction, Soxhlet extraction, and pressurized liquid extraction). Among the investigated solvents, ethanol provided the best fucoxanthin extraction yield (15.71 mg/g freeze-dried sample weight). Fucoxanthin content in the extracts produced by the different methods was quite constant (15.42–16.51 mg/g freeze-dried sample weight) but increased steeply based on the percentage of ethanol in water, emphasizing the importance of ethanol in the extraction. The results indicate that P. tricornutum is a rich source of fucoxanthin (at least ten times more abundant than that in macroalgae) that is easily extracted with ethanol, suggesting potential applications in human and animal food, health, and cosmetics.     

Direct injection horse urine analysis for the quantification and identification of threshold substances for doping control. III. Determination of salicylic acid by liquid chromatography/quadrupole time-of-flight mass spectrometry

In equine sport, salicylic acid is prohibited with a threshold level of 750 μg mL⁻¹ in urine; hence, doping control laboratories have to establish quantitative and qualitative methods for its determination. A simple and rapid liquid chromatographic/mass spectrometric method was developed and validated for the quantification and identification of salicylic acid. Urine samples after 900-fold dilution and addition of the internal standard (4-methylsalicylic acid) were directly injected to the liquid chromatography/quadrupole time-of-flight mass spectrometry system. Electrospray ionization in negative mode with full scan acquisition mode and product ion scan mode were chosen for the quantification and identification of salicylic acid, respectively. Run time was 2.0 min. The tested linear range was 2.5–50 μg mL⁻¹ (after 100-fold sample dilution). The relative standard deviations of intra- and inter-assay analysis of salicylic acid in horse urine were lower than 2.5% and 2.8%, respectively. Overall accuracy (relative percentage error) was less than 3.3%. Method was applied to two real samples found to be positive for salicylic acid, demonstrating simplicity, accuracy, and selectivity.     

Comprehensive ultra-high pressure capillary liquid chromatography/ion mobility spectrometry

Summary An ultra-high pressure liquid chromatograph was interfaced to a moderately high-resolution nanoelectrospray ionization ion mobility spectrometer operating at ambient temperature and pressure to achieve fast multidimensional separations. The potential of using ion mobility spectrometry as the second dimension in a comprehensive arrangement with liquid chromatography to offer improved qualitative information for compounds under specific operating conditions, is discussed. Separation and detection of selected benzodiazepines and triazine herbicides are demonstrated. Composite peak capacities of 39 and 33 for benzodiazepine and triazine herbicide mixtures, respectively, were achieved in less than 75 s using a 16.5 cm×50 μm internal diameter fused silica capillary liquid chromatographic column packed with 1.5 μm diameter ODS-bonded silica particles.     

Fast liquid chromatography/tandem mass spectrometry (highly selective selected reaction monitoring) for the determination of toltrazuril and its metabolites in food

In this work a fast liquid chromatography (LC)–tandem mass spectrometry (MS/MS) method was developed for the analysis of toltrazuril, a coccidiostatic drug, and its metabolites in meat food products. The applicability of atmospheric pressure chemical ionization (APCI) and heated electrospray ionization in both positive and negative modes was studied. APCI in negative mode provided the best results and the base peak originated from the loss of CF₃ (toltrazuril and toltrazuril sulfone) and CHF₃• (toltrazuril sulfoxide) was used as the precursor ion in MS/MS. A fast LC separation on a C₁₈ Fused-Core™ column was used together with the APCI-MS/MS method developed using enhanced mass resolution mode (highly selective selected reaction monitoring, H-SRM) to improve the sensitivity and selectivity for the analysis of these compounds in food samples. A simple sample treatment based on an extraction with acetonitrile and a cleanup with a C₁₈ cartridge was used. The LC-MS/MS (H-SRM) method showed good precision (relative standard deviation lower than 10%), accuracy, and linearity and allowed the determination of these compounds in food samples down to the parts per billion level (limits of detection between 0.5 and 5 μg kg^-1).     

Semi-automated liquid chromatography-mass spectrometry (LC-MS/MS) method for basic pesticides in wastewater effluents

Effluent from wastewater treatment plants have been identified as an important source of micro-organic contaminants in the environment. An online high-performance liquid chromatography–heated electrospray ionization tandem mass spectrometric method was developed and validated for the determination of basic pesticides in effluent wastewaters. Most available methods for pesticide analysis of wastewater samples are time-consuming, require complex clean-up steps and are difficult to automate. The method developed used a simple solid-phase extraction clean-up for salt and lipid reduction. On-line sample pre-concentration was performed using a reversed phase (C₁₈) column, and analytes were separated by back-flushing onto an analytical column (C₈) with detection using QqQ MS. An option to increase MS resolution was exploited to minimize interference from endogenous compounds in the matrix. A better than unit mass resolution was used (Q1 full width half maximum (FWHM) = 0.2 Da and Q3 FWHM = 0.7 Da), which was as rugged as a unit resolution method, and improved signal/noise and better detection limits were achieved for the targeted basic pesticides. This method was applied to the determination of 11 pesticides, including methoxytriazine, chlorotriazines, chloroacetanilides, phenylurea and carbamate pesticides. The percentage recovery values for these pesticides using the online trapping column were within the range, 73–95%, with relative standard deviation (RSD) values <8.9%. The highest concentrations of these pesticides in wastewater effluents in County Cork, Ireland, were simazine (0.51 μg/L), prometon (0.14 μg/L), diuron (0.21 μg/L) and atrazine (0.19 μg/L).     

Development and validation of a method for determination of residues of 15 pyrethroids and two metabolites of dithiocarbamates in foods by ultra-performance liquid chromatography-tandem mass spectrometry

This paper reports a novel approach for the detection, confirmation, and quantification of 15 selected pyrethroid pesticides, including pyrethins, and two metabolites of dithiocarbamates in foods by ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS–MS). The proposed method makes use of a modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) procedure that combines isolation of the pesticides and sample cleanup in a single step. Analysis of pyrethroids and dithiocarbamate metabolites was performed by UPLC–MS–MS operated with electrospray and atmospheric pressure chemical ionization, respectively. Two specific precursor–product ion transitions were acquired per target compound in multiple reaction monitoring (MRM) mode. Such acquisition achieved the minimum number of identification points according to European Commission (EC) document no. SANCO/10684/2009, thus fulfilling the EC point system requirement for identification of contaminants in samples. The method was validated with a variety of food samples. Calibration curves were linear and covered from 1 to 800 μg kg⁻¹ in the sample for all target compounds. Average recoveries, measured at mass fractions of 10 and 100 μg kg⁻¹ for pyrethroids and 5 and 50 μg kg⁻¹ for dithiocarbamate metabolites, were in the range of 70–120% for all target compounds with relative standard deviations below 20%. Method limits of quantification (MLOQ) were 10 μg kg⁻¹ and 5 μg kg⁻¹ for pyrethroids and dithiocarbamate metabolites, respectively. The method has been successfully applied to the analysis of 600 food samples in the course of the first Hong Kong total diet study with pyrethroids and metabolites of dithiocarbamates being the pesticides determined.     

Determination of the products of the oxidative transformation of unsymmetrical dimethylhydrazine in soils by liquid chromatography/mass spectrometry

Procedures are developed on the basis of liquid chromatography/mass spectrometry for determining the transformation products of unsymmetrical dimethylhydrazine in soils: formic acid dimethylhydrazide (1-formyl-2,2-dimethylhydrazine, analytical range 0.01–20 mg/kg), 1-methyl-1,2,4-triazole (analytical range 0.05–100 mg/kg), 1,1-dimethylguanidine (analytical range 0.05–100 mg/kg), and dimethylamine (analytical range 0.25–250 mg/kg). The measurements were performed in the mode of chemical ionization under atmospheric pressure followed by the registration of positive ions corresponding to the protonated forms of the components to be determined. A version of ion chromatography was elaborated for separation. For sample pretreatment, the use of extraction with methanol (for 1-formyl-2,2-dimethylhydrazine) and ultrasonic extraction with a weakly alkaline buffer solution (for other substances) was proposed.     

A sensitive liquid chromatographic-mass spectrometric method for simultaneous determination of dehydroevodiamine and limonin from Evodia rutaecarpa in rat plasma

A liquid chromatographic–mass spectrometric (LC–MS) method has been developed and validated for simultaneous determination of dehydroevodiamine and limonin from Evodia rutaecarpa in rat plasma. After addition of the internal standard, domperidone, plasma samples were extracted by liquid–liquid extraction with ethyl acetate and separated on an Apollo C₁₈ column (250 mm × 4.6 mm, 5 μm), with methanol–0.01% formic acid water (60:40, v/v) as mobile phase, within a runtime of 12.0 min. The analytes were detected without interference in the selected ion monitoring (SIM) mode with positive electrospray ionization. The linear range was 1.0–500 ng mL⁻¹ for dehydroevodiamine and 2.0–1,000 ng mL⁻¹ for limonin, with lower limits of quantitation of 1.0 and 2.0 ng mL⁻¹, respectively. Intra-day and inter-day precision were within 6.0% and 10.9%, respectively, for both analytes, and the accuracy (relative error, RE, %) was less than 4.8% and 6.5%, respectively. The validated method was successfully applied to a comparative pharmacokinetic study of dehydroevodiamine and limonin in rat plasma after oral administration of dehydroevodiamine, limonin, and an aqueous extract of Evodiae fructus. The results indicated there were obvious differences between the pharmacokinetic behavior after oral administration of an aqueous extract of Evodiae fructus compared with single substances.