Quantification of peptides using N-terminal isotope coding and C-terminal derivatization for sensitive analysis by micro liquid chromatography-tandem mass spectrometry.

Stable isotope-coding coupled with mass spectrometry is a popular method for quantitative proteomics and peptide quantification. However, the efficiency of the derivatization reaction at a particular functional group, especially in complex structures, can affect accuracy. Here, we present a dual functional-group derivatization of bioactive peptides followed by micro liquid chromatography-tandem mass spectrometry (LC-MS/MS). By separating the sensitivity-enhancement and isotope-coding derivatization reactions, suitable chemistries can be chosen. The peptide amino groups were reductively alkylated with acetaldehyde or acetaldehyde-d4 to afford N-alkylated products with different masses. This process is simple, quick and high-yield, and accurate comparative analysis can be achieved for the mass-differentiated peptides. Then, the carboxyl groups were derivatized with 1-(2-pyrimidinyl)piperazine to increase MS/MS sensitivity. Angiotensins I-IV, bradykinin and neurotensin were analyzed after online solid phase extraction by micro LC-MS/MS. In all instances, a greater than 17-fold increase in sensitivity was achieved, compared with the analyses of the underivatized peptides. Furthermore, the values obtained from the present method were in agreement with the result from isotope dilution quantification using isotopically labeled angiotensin I [Asp-Arg-(Val-d8 )-Tyr-Ile-His-Pro-(Phe-d8 )-His-Leu]. Copyright © 2016 John Wiley & Sons, Ltd.

Systematic evaluation of commercially available ultra-high performance liquid chromatography columns for drug metabolite profiling: optimization of chromatographic peak capacity.

The present study investigated the practical use of modern ultra-high performance liquid chromatography (UHPLC) separation techniques for drug metabolite profiling, aiming to develop a widely applicable, high-throughput, easy-to-use chromatographic method, with a high chromatographic resolution to accommodate simultaneous qualitative and quantitative analysis of small-molecule drugs and metabolites in biological matrices. To this end, first the UHPLC system volume and variance were evaluated. Then, a mixture of 17 drugs and various metabolites (molecular mass of 151-749Da, logP of -1.04 to 6.7), was injected on six sub-2µm particle columns. Five newest generation core shell technology columns were compared and tested against one column packed with porous particles. Two aqueous (pH 2.7 and 6.8) and two organic mobile phases were evaluated, first with the same flow and temperature and subsequently at each column's individual limit of temperature and pressure. The results demonstrated that pre-column dead volume had negligible influence on the peak capacity and shape. In contrast, a decrease in post-column volume of 57% resulted in a substantial (47%) increase in median peak capacity and significantly improved peak shape. When the various combinations of stationary and mobile phases were used at the same flow rate (0.5mL/min) and temperature (45°C), limited differences were observed between the median peak capacities, with a maximum of 26%. At higher flow though (up to 0.9mL/min), a maximum difference of almost 40% in median peak capacity was found between columns. The finally selected combination of solid-core particle column and mobile phase composition was chosen for its selectivity, peak capacity, wide applicability and peak shape. The developed method was applied to rat hepatocyte samples incubated with the drug buspirone and demonstrated to provide a similar chromatographic resolution, but a 6 times higher signal-to-noise ratio than a more traditional UHPLC metabolite profiling method using a fully porous particle packed column, within one third of the analysis time. In conclusion, a widely applicable, selective and fast chromatographic method was developed that can be applied to perform drug metabolite profiling in the timeframe of a quantitative analysis. It is envisioned that this method will in future be used for simultaneous qualitative and quantitative analysis and can therefore be considered a first important step in the Quan/Qual workflow.

Performance assessment of microflow LC combined with high-resolution MS in bioanalysis.

BACKGROUND: There continues to be consistent pressure for bioanalytical scientists to achieve lower limits of quantitation. The reasons range from smaller sample volumes available for analysis, to more potent analytes and the growth of biologics in drug development. This has led scientists to investigate alternative LC techniques, including microflow and nanoflow. These techniques have been shown to increase sensitivity of electrospray methods and reduce ionization matrix effects. Because high-resolution MS has significant benefits for the analysis of biologics, this type of mass spectrometer is becoming increasingly important in bioanalysis. RESULTS: For microflow analysis, a new ion source and significant extra sample preparation or chromatographic separation are not required. However, increased sensitivity and reduced matrix effects were consistently demonstrated when compared with UHPLC flow rates. The extent of matrix effects observed were compound dependent. DISCUSSION: This paper presents the utility of combining high-resolution/accurate mass with microflow LC from a quantitative standpoint. This includes evaluating the typical quantitative parameters of sensitivity, linearity/dynamic range, precision and accuracy. It also includes the evaluation of changes in signal suppression using microflow LC and microspray ionization. The benefits and disadvantages of using the combination of these two technologies for quantitative bioanalysis are also discussed.

The exposure of highly toxic aconitine does not significantly impact the activity and expression of cytochrome P450 3A in rats determined by a novel ultra performance liquid chromatography-tandem mass spectrometric method of a specific probe buspirone.

Aconitum species are widely used to treat rheumatism, cardiovascular diseases, and tumors in China and other Asian countries. The herbs are always used with drugs such as paclitaxel. Aconitine (AC) is one of the main bioactive/high-toxic alkaloids of Aconitum roots. AC is metabolized by cytochrome P450 (CYP) 3A. However, whether AC inhibits/induces CYP3A, which causes drug-drug interaction (DDI) is unclear. Our study aims to explore the potent effects of AC, as a marker component of Aconitum, on CYP3A using the probe buspirone in rats. The effects of oral AC on pharmacokinetics of buspirone were evaluated. CYP3A activity and protein levels in rat liver microsomes pretreated with oral AC were also measured using in vitro buspirone metabolism and Western blot. Buspirone and its major metabolites 1-(2-pyrimidinyl)piperazine and 6'-hydroxybuspirone were determined using a newly validated UPLC-MS/MS method. Single dose and 7-day AC administration at 0.125mg/kg had no effect on CYP3A activity since no change in the formation of 1-(2-pyrimidinyl)piperazine and 6'-hydroxybuspirone. CYP3A activity and protein levels in liver microsomes were also not affected by 7-day AC pretreatment at 0.125mg/kg. Therefore, AC neither inhibits nor induces CYP3A in rats, indicating AC does not cause CYP3A-related DDI in the liver.

A semi-automated method for the integrated evaluation of half-life and metabolic soft spots of discovery compounds.

BACKGROUND: An integrated method that provides rates of both parent disappearance and metabolite formation was developed. RESULTS: Buspirone, mirtazapine and verapamil were used as model compounds in developing the method. Incubations were carried out on a robotic platform. Qualitative analysis of metabolites in 30 µM samples was conducted by data-dependent HPLC-MS/MS on a high-resolution instrument. Quantitative analysis of the parent compound and metabolites in 0.5 µM samples was conducted by full-scan MS(2) with product ion extraction using an ion trap mass spectrometer. Data generated for the compounds included half-life and intrinsic clearance of the parent molecule, characterization of metabolites and relative rates of metabolite formation. A correction factor was used to convert MS responses of metabolites in 0.5 µM samples to UV areas in order to compare relative metabolite concentrations. CONCLUSION: The approach allows for the investigation of a set of six compounds simultaneously, with a turnaround time of 1 week or less.

Quantitative analysis of atractylenolide I in rat plasma by LC-MS/MS method and its application to pharmacokinetic study.

A new high-performance liquid chromatography/tandem mass spectrometry (LC-MS/MS) was developed for quantitative analysis of atractylenolide I in rat plasma using buspirone as internal standard (I.S.). Rat plasma samples were deproteined with methanol and acetonitrile (1:1, v/v). Atractylenolide I and I.S. were separated on a Phenomenex Gemini C(18) column (50 mm × 2.0 mm, 5 µm) with gradient mobile phase at the flow rate of 0.4 ml/min. The detection was performed by positive ion electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode. The linear calibration curve of atractylenolide I in rat plasma ranged 2.0-5000 ng/ml (R>0.9979). The limit of detection (LOD) and the limit of quantification (LOQ) were 0.6 ng/ml and 2.0 ng/ml, respectively. Both accuracy and precision of the assay were satisfactory. The recoveries of atractylenolide I and I.S. were 91.4% and 87.8%, respectively. This fully validated method was applied to a pharmacokinetic study of atractylenolide I in rats administered with 20 g/kg Atractylodis extract. The main pharmacokinetic parameters T(max) (the time to peak), C(max) (the concentration to peak), T(0.5) (the biological half time), and K(e) (the elimination rate constant) were 0.81 ± 0.11h, 7.99 ± 1.2 ng/ml, 1.94 ± 0.27 h, 0.365 ± 0.06/h, respectively.

Buspirone, fexofenadine, and omeprazole: quantification of probe drugs and their metabolites in human plasma.

Probe drugs are critical tools for the measurement of drug metabolism and transport activities in human subjects. Often several probe drugs are administered simultaneously in a "cocktail". This cocktail approach requires efficient analytical methods for the simultaneous quantitation of multiple analytes. We have developed and validated a liquid chromatography-tandem mass spectrometry method for the simultaneous determination of three probe drugs and their metabolites in human plasma. The analytes include omeprazole and its metabolites omeprazole sulfone and 5'-hydroxyomeprazole; buspirone and its metabolite 1-[2-pyrimidyl]-piperazine (1PP); and fexofenadine. These analytes and the internal standard lansoprazole were extracted from plasma using protein precipitation with acetonitrile. Gradient reverse-phase chromatography was performed with 7.5mM ammonium bicarbonate and acetonitrile, and the analytes were quantified in positive ion electrospray mode with multiple reaction monitoring. The method was validated to quantify the concentration ranges of 1.0-1000ng/ml for omeprazole, omeprazole sulfone, 5'-hydroxyomeprazole, and fexofenadine; 0.1-100ng/ml for buspirone, and 1.0-100ng/ml for 1PP. These linear ranges span the plasma concentrations for all of the analytes from probe drug studies. The intra-day precision was between 2.1 and 16.1%, and the accuracy ranged from 86 to 115% for all analytes. Inter-day precision and accuracy ranged from 0.3 to 14% and from 90 to 110%, respectively. The lower limits of quantification were 0.1ng/ml for buspirone and 1ng/ml for all other analytes. This method provides a fast, sensitive, and selective analytical tool for quantification of the six analytes in plasma necessary to support the use of this probe drug cocktail in clinical studies.

Spectroscopic study of the reaction mechanism of buspirone interaction with iodine and tetracyanoethylene reagents and its applications.

The reactions between the drug buspirone (busp) in its base form and iodine amphoteric reagent (n-donor and/or sigma-acceptor) and with tetracyanoethylene as a pi-acceptor reagent (TCNE) have been studied spectrophotometrically at different reactant concentrations, time intervals, temperatures, and with different solvents and wavelengths, with the aim of selecting the conditions that give the most suitable molar extinction coefficients. This study aims chiefly to throw light on the nature of these reactions and to select the most proper conditions for spectrophotometric application of these reagents to determine this biologically active drug used in treating different diseases. The reaction mechanism involves the formation of busp-I(2) outer and inner sphere complexes. The separated busp-I(2) solid product obtained was investigated using elemental analyses, FT-IR, thermal analyses (TA) and electron ionization mass spectrometry (EI-MS) and was found to be biologically active. The reaction mechanism of busp-TCNE involves the formation of a charge transfer (CT) complex. The analytical parameters of the proposed spectrophotometric procedures were calculated. These procedures were applied in the analysis of busp in its formulations as a drug used to treat psychiatric illnesses. The values of the Sandell sensitivity, standard deviation (SD), relative standard deviation (RSD) and recovery percentage show the high sensitivity of these procedures. This study also presents a promising new busp-I(2) drug derivative that can be used more efficiently for the same purposes as its parent. It gives a clear idea about the possible metabolites and metabolic pathways of busp and its derivative that may occur in vivo.

Flow injection analysis with tubular membrane ion-selective electrode and coated wires for buspirone hydrochloride.

New Plastic membrane ion-selective electrode for buspirone hydrochloride based on buspironium tetraphenylborate was prepared. The electrode exhibited mean slope of calibration graph of 58.4 mV per decade of BusCl concentration at 25 degrees C. The electrode can be used within the concentration range 6.3 x 10(-5) - 10(-2) M BusCl at a pH range of 2.5-7.0. The standard electrode potentials were determined at different temperatures and used to calculate the isothermal temperature coefficient of the electrode, amounting to 0.00056 V degrees C(-1). The electrode showed a very good selectivity for BusCl with respect to a number of inorganic cations, sugars and amino acids. The electrode was applied to the potentiometric determination of the buspirone ion and its pharmaceutical preparation under batch and flow injection conditions. Also, buspirone was determined by conductimetric titrations. Graphite rod, copper and silver coated wire electrodes were prepared and characterized as sensors for the drug under investigation.

Simultaneous determination of metabolic stability and identification of buspirone metabolites using multiple column fast liquid chromatography time-of-flight mass spectrometry.

A recent trend in the drug discovery and development process is to shift the starting point of drug metabolism and pharmacokinetic (DMPK) studies to a time as early as possible in the development chain to address potential issues in parallel with the optimization of the drug's lead structure. Therefore, it is necessary to develop assay methods to determine early adsorption, distribution, metabolism and excretion (ADME) parameters like metabolic stability and metabolite identification. For metabolite identification it is of crucial importance to work with fast liquid chromatography/mass spectrometry (LC/MS) systems, which provide the necessary high throughput functionalities to handle a large number of samples in combination with high speed and high resolution chromatography as well as mass accuracy. In this study a fast two-column liquid chromatography (LC) method will be used to simultaneously determine metabolic stability and to identify metabolites of buspirone using highly accurate mass measurement by means of an electrospray time-of-flight (ESI-TOF) mass spectrometer. Whereby, the metabolic stability will be determined on a short sub-two micron column, the main metabolites will be identified in the same experiment by the automated use of a long sub-two micron column, which provides the necessary high resolution.

Hybrid triple quadrupole-linear ion trap mass spectrometry in fragmentation mechanism studies: application to structure elucidation of buspirone and one of its metabolites.

The use of a hybrid triple quadrupole-linear ion trap (QqQ(LIT)) mass spectrometer system for a comprehensive study of fragmentation mechanisms is described. The anxiolytic drug, buspirone, was chosen as a model compound for this study. With the advent of a QqQ(LIT) instrument, both the traditional quadrupole and the new linear ion trap scans (LIT) could be performed in a single LC run. In the past, a sample had to be run on two different instruments, namely, a triple quadrupole instrument (QqQ) and a 3D ion trap (3D IT) to obtain similar information. With the new QqQ(LIT) technology, collision-induced dissociation (CID) occur in a quadrupole collision cell, q2, and fragment ions are trapped and analyzed in Q3 operated in LIT mode. In this work, high-sensitivity product ion spectra of buspirone were obtained from the one-stage 'Enhanced Product Ion' scan (EPI) with rich product ions and no low mass cut-off. Furthermore, detailed fragmentation pathways were elucidated by further dissociation of each of the fragment ions in the EPI spectrum using MS(3) mode in the same run. The MS(3) scan was performed by incorporating CID in q2, and trapping, cooling, isolation, and resonance-excitation in Q3 when operating in LIT mode. This approach allowed unambiguous assignment of all fragment ions quickly with fewer experiments and easier interpretation than the previous approach. The overall sensitivity for obtaining complete fragment ion data was significantly improved for QqQ(LIT) as compared with that of QqQ and 3D IT mass spectrometers. This is beneficial for structure determination of unknown trace components. The method allowed structure determination of metabolites of buspirone in rat microsomes at 1 microM concentration, which was a 10-fold lower concentration than was needed for QqQ or 3D IT instruments. The QqQ(LIT) instrument provided a simple, rapid, sensitive and powerful approach for structure elucidation of trace components.

Analysis of low level radioactive metabolites in biological fluids using high-performance liquid chromatography with microplate scintillation counting: method validation and application.

TopCount, a microplate scintillation counter (MSC), has been recently employed as an off-line liquid radiochromatographic detector for radioactive metabolite profile analysis. The present study was undertaken to validate TopCount for metabolite profiling with respect to sensitivity, accuracy, precision and radioactivity recovery. Matrix effects of various human samples on TopCount performance and capability of MSC for volatile metabolite analysis were also investigated. TopCount had a limit of detection (LOD) of 5 DPM and a limit of quantification (LOQ) of 15 DPM for [(14)C]-labeled compounds at a 10min counting time. It was two-fold more sensitive than a liquid scintillation counter (LSC), and 50-100-fold more sensitive than a radioactivity flow detector (RFD). TopCount had comparable accuracy and precision to RFD, and comparable precision to LSC for determining relative abundance of metabolites. Human liver microsome incubation (up to 1 mL), plasma (up to 1 mL), urine (up to 2 mL) and feces (up to 50mg) had no significant quenching effects on TopCount performance. Benzoic acid, a volatile metabolite, was detected by TopCount, but not by Microbeta counter after microplates were dried under vacuum. Radioactivity recovery in HPLC-MSC analysis was reliably determined using an LSC-based method. Examples of using HPLC-MSC for analysis of low levels of radioactive metabolites are presented, including determination of plasma metabolite profile, in vitro reactive metabolites trapped by [(3)H]glutathione, and metabolite concentrations in an enzyme kinetic experiment. The data from this study strongly suggest that HPLC in combination with TopCount is a viable alternative analytical tool for detection and quantification of low levels of radioactive metabolites in biological fluids.

Adsorptive stripping voltammetric behaviour of azomethine group in pyrimidine-containing drugs.

The stripping voltammetric behaviour of buspirone hydrochloride (BUS) and piribedil (PIR), as models of pyrimidine-containing compounds, was studied using a hanging mercury drop electrode (HMDE). A sensitive adsorptive stripping voltammetric method for determination of such drugs is described. The voltammetric peaks were obtained at -1.23 and -1.22 V for BUS and PIR. respectively, which correspond to the reduction of the azomethine group of pyrimidine ring in Britton-Robinson buffer (pH 7). Factors such as pH of supporting electrolyte, accumulation potential and time and instrumental parameters were optimized. Calibration plots and regression data validation, accuracy, precision, limits of detection, limits of quantification, and other aspects of analytical merit are presented. The applicability of the method was evaluated through determination of BUS and PIR in tablet dosage forms. A preliminary study of the analysis of plasma samples, spiked with the investigated drug, after a simple extraction procedure is described.

Development and validation of a high-performance liquid chromatographic method for the determination of buspirone in pharmaceutical preparations.

A stability indicating, reversed-phase high-performance liquid chromatographic method was developed and validated for the determination of buspirone (Bsp) in pharmaceutical dosage forms. The use of a semi-micro XTerra MS C18 (150 mm x 3.0 mm i.d., 5 microm particle size) analytical column, results in substantial reduction in solvent consumption and increased sensitivity. The mobile phase consisted of a mixture of 0.010 M ammonium acetate (pH 4.0) and methanol (55:45, v/v), pumped at a flow rate 0.30 ml min-1. The UV detector was operated at 245 nm. The retention times for lidocaine (Ldc), which was used as internal standard, and buspirone were 4.57 and 7.72 min, respectively. The calibration graph was ranged from 1.00 to 5.00 microg ml-1, while detection and quantitation limits were found to be 0.22 and 0.67 microg ml-1, respectively. The intra- and inter-day relative standard deviation (% R.S.D.) values were less than 1.94%, while the relative percentage error (% Er) was less than 4.0% (n = 5). The method was applied to the quality control of commercial tablets and content uniformity test and proved to be suitable for rapid and reliable quality control.

Electrospray mass spectrometric studies of noncovalent complexes of buspirone hydrochloride and other serotonin 5-HT(1A) receptor ligands containing arylpiperazine moieties.

Noncovalent complexes consisting of two protonated amines and a chloride anion were observed under electrospray ionization mass spectrometry (ESI-MS) conditions. The observed phenomenon was investigated for the hydrochlorides of buspirone, a well-known anxiolytic drug, and 23 other arylpiperazine derivatives that had been developed as serotonin 5-HT(1A) receptor ligands. Due to the major role of ionic interactions in a vacuum, it was proposed that the detected complexes were formed by NH(+)---Cl(-)---NH(+) bridges. It was found that complexation depended on structural features of the analyzed compounds. For derivatives with a shorter linker (three methylene groups) containing a terminal cyclic amide fragment, complex ions were not observed. It was postulated that, in the latter case, steric hindrance due to a terminal group could disturb ionic bridge formation. Since both the observed complexation and ligand-binding processes are driven by noncovalent forces, and a qualitative relationship between them was found (compounds with a 4-carbon chain always display higher affinity for 5-HT(1A) receptors than do their 3-carbon analogues), such ESI-MS studies may yield valuable information on ligand-receptor interactions.

Liquid chromatographic method for the analysis of buspirone HCl and its potential impurities.

An accurate, reproducible, and sensitive method for the determination of buspirone HCl and its potential impurities is developed and validated. The validated liquid chromaography method is conducted to meet the Food and Drug Administration/ International Conference on Harmonization requirements for the analysis of buspirone HCI in the presence of its impurities. Five buspirone HCI potential impurities, including 1-(2-pyrimidinyl)-piperazine (I), propargyl chloride (II), 3,3'-tetramethylene glutarimide (III), propargyl glutarimide (IV), and the Mannich base-condensate of I-IV fumarate (V), are separated using a microBondapack C18 column by gradient elution with a flow rate 2.0 mL/min. The initial mobile phase composition is 90:10 (v/v) 10mM KH2PO4 (pH 6.1)-acetonitrile. After a 1-min initial hold, a linear gradient is performed in 26 min to 35:65 (v/v) 10mM KH2PO4 (pH 6.1)-acetonitrile. The samples are detected at 210 and 240 nm using a photo-diode array detector. The linear range of detection for buspirone HCI was between 1.25 ng/microL and 500 ng/microL, with a limit of quantification of 1.25 ng/microL. The linearity, range, peak purity, selectivity, system performance parameters, precision, accuracy, and robustness for all of the impurities were also shown to have acceptable values.

Stability-indicating high-performance liquid chromatographic assay of buspirone HCl.

The United States Pharmacopoeia high-performance liquid chromatographic (HPLC) assay method of buspirone is not able to discriminate buspirone from its degradation products. The purpose of this work is to develop a sensitive, selective, and validated stability-indicating HPLC assay for the analysis of a buspirone hydrochloride in a bulk drug. Buspirone HCI and its potential impurities and degradation products are analyzed on an Ultrasphere C18 column heated to 40 degrees C using a gradient program that contains monobasic potassium phosphate buffer solution (pH 6.9) and acetonitrile-methanol mixture (13:17) of 35% for 5 minutes, then increased to 54% in 5.5 minutes. The samples are monitored using a photo-diode array detector and integrated at 244 and 210 nm. The stress testing of buspirone HCI shows that buspirone acid hydrochloride is the major degradation product. The developed method shows a separation of buspirone degradation product and its potential impurities in one run. The stability of buspirone HCI is studied under accelerated conditions in order to provide a rapid indication of differences that might result from a change in the manufacturing process or source of the sample. The forced degradation conditions include the effect of heat, moisture, light, acid-base hydrolysis, sonication, and oxidation. The compatibility of buspirone HCI with some pharmaceutical excipients is studied under stress conditions. The linear range of buspirone HCI is between 5 and 200 ng/microL with a limit of quantitation of 2.5 ng/microL. The intraassay percentage deviation is not more than 0.38%, and the day-to-day variation was not more than 0.80%. The selectivity, repeatability, linearity, range, accuracy, sample solution stability, ruggedness, and robustness show acceptable values.

Measurement and pharmacokinetic analysis of buspirone by means of brain microdialysis coupled to high-performance liquid chromatography with electrochemical detection.

The feasibility of an electrochemical detection system with on-line microdialysis coupled with sensitive microbore high-performance liquid chromatography for the measurement and brain pharmacokinetic analysis of buspirone was investigated. A microdialysis probe was inserted into the right striatum of male Sprague-Dawley rats, which had been administered buspirone 10 mg/kg. i.v.). Dialysates were automatically injected through an on-line injector into a cyano microbore column coupled to an electrochemical detector. Samples were eluted with a mobile phase containing 0.1 M monosodium dihydrogenphosphate acetonitrile-diethylamine (85:15:0.1, v/v/v). pH 3.0, adjusted with orthophosphoric acids at a flow-rate of 0.06 ml/min. A biphasic phenomenon with a rapid distribution phase followed by a slower elimination phase was observed from the brain buspirone concentration-time curve. The results indicate that the brain pharmacokinetics of buspirone appear to conform to a two-compartment model.

Rapid, sensitive procedure to determine buspirone levels in rat brains using gas chromatography with nitrogen-phosphorus detection.

Reported here is a rapid, sensitive and relatively inexpensive procedure using gas chromatography with nitrogen-phosphorus detection (GC-NPD) to quantify buspirone levels in brains of rats. The analyte was directly extracted from brain homogenate with toluene after basification and then subjected to GC-NPD analysis using a capillary column. The calibration curves were linear over the range of 10 to 320 ng per 2 ml of brain homogenate, with typical r2 values >0.99. The assay was highly reproducible and gave peaks with excellent chromatographic properties.

Liquid chromatography/chemical reaction interface mass spectrometry as an alternative to radioisotopes for quantitative drug metabolism studies.

Chemical reaction interface mass spectrometry (CRIMS) was coupled on-line with HPLC using a Vestec particle beam interface. A helium-assisted nebulizer provided added stability with no loss in accuracy or precision as compared to the thermospray nebulizer at flow rates of up to 1.0 mL/min using isocratic conditions. However, mass spectral response was found to be solvent-dependent for both the helium-assisted and thermospray nebulizers. Postcolumn solvent addition of methanol eliminated solvent-dependent decreases in mass spectral response. This allowed gradient HPLC elutions to be performed. Under these conditions, the flow of solvent into the particle beam interface was 2.5 mL/min, so a conventional thermospray nebulizer had to be used instead of the helium-assisted nebulizer. Experiments were conducted with the antianxiety agent buspirone in order to validate the methodology. Metabolites from in vitro incubations of [15N]/[14C]buspirone with rat liver slices were analyzed by gradient LC/CRIMS and by gradient LC/[14C] radioactivity counting. The response from LC/CRIMS analysis for individual metabolites was then compared with that obtained by LC/[14C] radioactivity counting. An excellent correlation was observed between the two methods for metabolites with quite different HPLC characteristics. Thus, gradient LC/CRIMS in combination with stable isotopes provides an alternative to using radioisotopes for carrying out drug metabolism studies.