Chemiluminescent Nitrogen Detector Research Articles

Quality Control in Combinatorial Chemistry: Determinations of Amounts and Comparison of the “Purity” of LC−MS-Purified Samples by NMR, LC−UV and CLND

The absolute purities of 20 purified samples from a combinatorial library have been determined by a new method that uses the DMSO sidebands [1J[13C-1H]] as an internal standard for quantification. The obtained absolute amounts are compared with the amounts of sample obtained by weighing, with the calculated weights obtained by chemiluminescent nitrogen detection (CLND) chromatography and with the relative purities obtained by LC-UV chromatography.

Concentration Determination of a Recombinant Vaccine Antigen Adsorbed onto an Alum Adjuvant by Chemiluminescent Nitrogen Detection

A chemiluminescent nitrogen detector (CLND) has been evaluated for determining the concentration of an aluminum-adsorbed recombinant vaccine antigen. Quantification of the antigen was based upon several nitrogen-containing compounds used to calibrate the CLND. All calibrants (6.75-400 microg/ml) generated linear standard curves, with slopes being directly proportional to the % nitrogen. The limit of quantification (LOQ) was determined to be 6.75 microg/ml based on the performance of the antigen standard curve, and the limit of detection (LOD) was defined by setting the CLND minimum peak area to 40,000 U. The CLND was capable of analyzing antigen-adjuvant suspensions (adsorbed + unbound antigen) without any sample pretreatment. To measure unbound antigen, the suspension was centrifuged and an aliquot of supernatant removed for analysis; the difference between these two measurements was the amount of adsorbed antigen. The adjuvant exhibited no significant matrix effect. Samples were analyzed in triplicate with observed relative standard deviation values ranging from 0.065% to 10.0%. The most accurate concentrations of the antigen were recovered relative to the antigen itself and to glycine as standards. This methodology provides a direct measurement of the concentration of a vaccine antigen adsorbed onto an aluminum adjuvant.

Determination of topiramate and its degradation product in liquid oral solutions by high performance liquid chromatography with a chemiluminescent nitrogen detector

Topiramate is a sulfamate-substituted monosaccharide that is prescribed for the treatment of epilepsy. It has been a challenge to develop analytical methods for topiramate formulations because the compounds of interest do not have chromophores that are active above 190 nm and because of interference from excipients. This paper describes a simple, specific, precise, accurate, and sensitive method using a chemiluminescent nitrogen detector. The method has a validated linearity range of 32–4800 ng of topiramate and excellent precision (system repeatability). The limit of quantitation was determined to be 0.1% for the degradation product w/w versus topiramate. The method has been successfully used for probe stability studies in support of early phase formulation development.

Streamlined System for Purifying and Quantifying a Diverse Library of Compounds and the Effect of Compound Concentration Measurements on the Accurate Interpretation of Biological Assay Results

As part of an overall systems approach to generating highly accurate screening data across large numbers of compounds and biological targets, we have developed and implemented streamlined methods for purifying and quantitating compounds at various stages of the screening process, coupled with automated "traditional" storage methods (DMSO, -20 degrees C). Specifically, all of the compounds in our druglike library are purified by LC/MS/UV and are then controlled for identity and concentration in their respective DMSO stock solutions by chemiluminescent nitrogen detection (CLND)/evaporative light scattering detection (ELSD) and MS/UV. In addition, the compound-buffer solutions used in the various biological assays are quantitated by LC/UV/CLND to determine the concentration of compound actually present during screening. Our results show that LC/UV/CLND/ELSD/MS is a widely applicable method that can be used to purify, quantitate, and identify most small organic molecules from compound libraries. The LC/UV/CLND technique is a simple and sensitive method that can be easily and cost-effectively employed to rapidly determine the concentrations of even small amounts of any N-containing compound in aqueous solution. We present data to establish error limits for concentration determination that are well within the overall variability of the screening process. This study demonstrates that there is a significant difference between the predicted amount of soluble compound from stock DMSO solutions following dilution into assay buffer and the actual amount present in assay buffer solutions, even at the low concentrations employed for the assays. We also demonstrate that knowledge of the concentrations of compounds to which the biological target is exposed is critical for accurate potency determinations. Accurate potency values are in turn particularly important for drug discovery, for understanding structure-activity relationships, and for building useful empirical models of protein-ligand interactions. Our new understanding of relative solubility demonstrates that most, if not all, decisions that are made in early discovery are based upon missing or inaccurate information. Finally, we demonstrate that careful control of compound handling and concentration, coupled with accurate assay methods, allows the use of both positive and negative data in analyzing screening data sets for structure-activity relationships that determine potency and selectivity.

Liquid chromatography–mass spectrometry with chemiluminescent nitrogen detection for on-line quantitative analysis of compound collections: advantages and limitations

Recently, alternative detection methods such as chemiluminescent nitrogen detection (CLND) have been coupled successfully with HPLC for quantification. This detector produces a signal proportional to the number of moles nitrogen present in the compound. Sample concentration of compounds with a known formula can be determined by use of an external calibration standard such as caffeine. Hence, the CLND can be used without the need for primary standards of the compound with unknown concentration, which enables the use of this detector for high-throughput analysis. In this work, the reliability and pitfalls of this coupled LC-MS/CLND are demonstrated. Nitrogen detection is specific as it only gives a response for nitrogen containing compounds and universal since it only gives a linear response. Nevertheless the lower response for N=N and N-N containing compounds has been evaluated in this study.

Liquid chromatography–mass spectrometry with chemiluminescent nitrogen detection for on-line quantitative analysis of compound collections: advantages and limitations

Recently, alternative detection methods such as chemiluminescent nitrogen detection (CLND) have been coupled successfully with HPLC for quantification. This detector produces a signal proportional to the number of moles nitrogen present in the compound. Sample concentration of compounds with a known formula can be determined by use of an external calibration standard such as caffeine. Hence, the CLND can be used without the need for primary standards of the compound with unknown concentration, which enables the use of this detector for high-throughput analysis. In this work, the reliability and pitfalls of this coupled LC–MS/CLND are demonstrated. Nitrogen detection is specific as it only gives a response for nitrogen containing compounds and universal since it only gives a linear response. Nevertheless the lower response for N N and N N containing compounds has been evaluated in this study.

Quantitation of drug metabolites in the absence of pure metabolite standards by high-performance liquid chromatography coupled with a chemiluminescence nitrogen detector and mass spectrometer.

Quantitative information on drug metabolites with pharmacological or toxicological activities is of great interest during the drug discovery and development process. Because the analyte response with mass spectrometry can change significantly due to small variations in chemical structure, pure standards are required to construct standard curves for quantitation. However, for most programs at the discovery stage, pure metabolite standards are not available. In this work, an evaluation was conducted using a chemiluminescent nitrogen detector (CLND) as a calibrator to obtain the response factor ratio on a mass spectrometer generated from a metabolite and its parent compound in biological fluids. Using the response factor ratio obtained from the CLND, the metabolite could be quantified with the liquid chromatography/tandem mass spectrometry (LC/MS/MS) response obtained from the parent drug's standard curve. For this evaluation, oxazepam and temazepam were chosen as a 'drug/metabolite' pair. Temazepam was treated as the methylated metabolite of oxazepam. A spiked dog urine sample with a known concentration of oxazepam and unknown concentration of temazepam was injected onto the HPLC system and detected by both the CLND and MS/MS. Taking advantage of the equimolar response feature of the CLND, a response factor ratio between temazepam and oxazepam on the mass spectrometer was obtained by comparing the peak areas generated on the CLND and the mass spectrometer. From this ratio, temazepam was quantified using the oxazepam standard curve. The difference between the concentration of temazepam obtained from the reconstructed standard curve and the concentration obtained directly from a real temazepam standard curve was within 13% except the least concentrated standard (31%). This methodology has been successfully applied to measure quantities of the metabolite of a proprietary compound in a dog pharmacokinetic (PK) study.

Speciation of nitrogen-containing compounds in diesel fuel by comprehensive two-dimensional gas chromatography.

Nitrogen-containing compounds in diesel fuel have been speciated by comprehensive two-dimensional gas chromatography with nitrogen chemiluminescence detector (GC x GC-NCD). The speciation of nitrogen-containing compounds in diesel is difficult because of low concentration and complexity. The advantages of GC x GC are improved resolution and enhanced sensitivity. GC x GC-NCD can achieve the type and class separation of nitrogen-containing compounds with an appropriate separation column combination. Diesel contains both neutral (indoles and carbazoles) and basic (pyridines and quinolines) nitrogen-containing compounds. Relative concentrations of each class as well as each carbon number family can be quantified by integrating their peak volumes. This study demonstrates the capability of GC x GC-NCD for speciation of nitrogen-containing compound classes.

Identification of BVT.2938 metabolites by LC/MS and LC/MS/MS after in vitro incubations with liver microsomes and hepatocytes

The metabolism of the 5HT2c agonist BVT.2938, 1-(3-{2-[(2-ethoxy-3-pyridinyl)oxy]ethoxy}-2-pyrazinyl)-2(R)-methylpiperazine, was studied in vitro by incubation with rat, monkey and human liver microsomes as well as cryopreserved hepatocytes, followed by liquid chromatography/mass spectrometry (LC/MS) and LC/MS/MS analysis on a quadrupole-time of flight mass spectrometer for structural elucidation. Deuterium exchange on column was used to differentiate between hydroxylation and N-oxidation. Liver microsomes were incubated in two different buffer systems with optimum conditions for cytochrome P450 activity or UDP-glucuronosyltransferase activity. The major phase I metabolites of BVT.2938 originated from O-deethylation of the pyridine ring, O-dealkylation of the ethylene bridge, pyrazine ring hydroxylation, hydroxylation of pyridine ring and piperazine ring N-hydroxylation. When a hydrogen carbonate buffer system was supplemented with UDPGA, the piperazine carbamoyl-glucuronide from the parent compound was identified together with several glucuronides of the phase I metabolites. The metabolite pattern in hepatocytes was similar to microsomes except that the sulphate at the N-position of the piperazine ring of BVT.2938 was identified, while the carbamoyl-glucuronide was missing. Excellent correlation was obtained between radioactivity detection and the chemiluminescent nitrogen detector when the nitrogen content of the analytes was taken into account.

Determination of airborne anhydrides using LC-MS monitoring negative ions of di-n-butylamine derivatives.

An air sampling method for simultaneous determination of organic acid anhydrides and isocyanates is presented. Air samples are collected in impinger flasks filled with 0.01 M di-n-butylamine (DBA) in a mixture of toluene-acetonitrile (7:3, v/v) with a 13 millimetre glass fibre filter in series. The amount of anhydrides and isocyanates are determined as their amide and urea derivatives using LC-MS. Four anhydrides, maleic anhydride (MA), phthalic anhydride (PA), tetrahydrophthalic anhydride (TA) and cis-hexahydrophthalic anhydride (HA) and 11 isocyanates could be separated in 9 minutes using gradient elution. Anhydride-DBA derivatives in standard solutions were quantified using LC with chemiluminescent nitrogen detection (CLND). Anhydride-DBA derivatives were found to be stable for at least two months when stored in acetonitrile or toluene in the freezer. The yield of DBA derivatives of anhydrides in the 0.01 M DBA in toluene-acetonitrile (7:3, v/v) was in the range of 70->95%. Using MS and negative electrospray ionisation (ES-) linear calibrations curves were obtained for the anhydrides with correlation coefficients ranging from 0.9970-0.9997. The instrumental detection limit for the anhydrides ranged from 10-30 fmol, based on a signal to noise root mean square (RMS) ratio of 3. Monitoring positive and negative ions simultaneously, both isocyanates and anhydrides could be determined as their DBA derivatives in the same chromatographic run. When air samples were collected during thermal degradation of different coated metal sheets both anhydrides and isocyanates were present in the same samples and all the studied anhydrides were found.

Accelerating the drug optimization process: identification, structure elucidation, and quantification of in vivo metabolites using stable isotopes with LC/MSn and the chemiluminescent nitrogen detector.

Most preclinical leads exhibit poor ADME/PK properties and require optimizing to increase the likelihood of becoming successful pharmaceuticals. As a means of accelerating the evaluation of these leads in vivo, we assessed the use of LC/MS with the chemiluminescent-nitrogen detector (CLND) and a stable isotope to identify and quantify in vivo metabolites and to measure excretion. A 14C-labeled preclinical lead that also contained two chlorine atoms was administered orally to rats, and samples of bile, urine, and plasma were collected and analyzed by LC with radiodetection and by LC/MS-CLND with the chlorine atoms used as tracers. Both methods identified seven metabolites in bile and two metabolites in urine. The amount and abundance of each metabolite was measured, and the results were equivalent for the two methods. Material balance was measured by liquid scintillation counting of the starting samples, by LC/radiodetection, and by LC/MS-CLND. All three methods yielded the same results and showed that the primary route of clearance was metabolism followed by immediate excretion. This study demonstrates that LC/MS-CLND with a stable isotope is a method that can efficiently track and accurately quantify metabolites, making it possible to rapidly study ADME/PK in vivo without radiolabeling.

A comparative study of commercial liquid chromatographic detectors for the analysis of underivatized amino acids

This study compares the main commercial detectors that can detect amino acids in their underivatized form. The detectors tested are: the chemiluminescent nitrogen detector (CLND), the evaporative light scattering detector (ELSD), the nuclear magnetic resonance spectrometer, conductivity detector, refractive index, UV, and electrospray quadrupole mass spectrometry (in simple and tandem MS mode). As ELSD, CLND and MS require a volatile mobile phase, an ion-pair reversed-phase liquid chromatographic system was selected, consisting of an octadecyl column and an aqueous mobile phase containing pentadecafluorooctanoic acid as volatile ion-pairing reagent. Underivatized taurine, hypotaurine, aspartic acid, hydroxyproline, asparagine, serine, glycine, glutamine, cysteine, glutamic acid, threonine and alanine were simultaneously analysed with each detector. In order to test the applicability of these detectors to “real world” samples, the amino acid stoichiometry of the tetrapeptide Gly–Gly–Asp–Ala was determined with each detector after acid hydrolysis. The detectors were compared in terms of linearity, limit of detection, advantages and disadvantages as well as special features (capacity to provide structural information, specificity, quantification with single calibration curve, etc.).

ALL-IN-ONE LIBRARY VALUATIONS

A NEW SYSTEM HAS BEEN DEveloped for evaluating, in a single experiment, the identity, purity, and concentration of combinatorial library compounds made by parallel synthesis. In the system, high-performance liquid chromatography (HPLC) separates components of synthetic samples that are arrayed on microtiter plates. The eluent is first analyzed by a UV-visible photodiode array (PDA) detector and is then directed to three other analyzers—an evaporative light-scattering detector (ELSD), a chemiluminescent nitrogen detector (CLND), and a time-of-flight mass spectrometry (TOFMS) unit. The approach was developed by analytical chemists David A. Yurek and Ming-Shang Kuo and information scientist Derek L. Branch of the Discovery Technologies unit at Pharmacia Corp. [ J. Comb. Chem. , 4 , 138 (2002)]. It was devised to solve a common parallel synthesis problem: Target compounds are produced in a range of yields and purities, making it difficult to screen them for biological activity. Scientists generally address th...

A capillary electrophoretic system with an improved gas-phase chemiluminescence nitrogen detector

A new, rugged nebulizer interface was designed to couple a capillary electrophoretic system to a gas-phase nitrogen chemiluminescence detector. By hydraulically decoupling the separation capillary from the nebulizer of the gas-phase nitrogen chemiluminescence detector, the interface-related loss of separation efficiency could be limited to a mere 10%. The ozone reaction chamber of the gas-phase nitrogen chemiluminescence detector has been redesigned leading to a threefold improvement in detection sensitivity over the old design. In combination with a field-amplified sample stacking procedure, the new system was used to detect 0.25 ppm concentrations of UV chromophore-free aminoglycosides.

Chemiluminescence nitrogen detection in ion chromatography for the determination of nitrogen-containing anions

Chemiluminescence nitrogen detection (CLND) provides equimolar response for nitrogen-containing ions such as nitrate, nitrite, cyanide, ammonium and tetradecyltrimethylammonium. Only azide yields a lower response. Nitrite, azide and nitrate are separated on a Dionex AS11 column using 5 m M NaOH as eluent with a 3 μ M (1 ng N) limit of detection. Matrices, such as 1:10 diluted seawater, do not degrade these detection limits. CLND also provides equally sensitive (limit of detection 3 μ M, 78 ppb) detection of weak acids such, as cyanide, which yield poor sensitivity with suppressed conductivity detection.

Quality assessment of combinatorial pooled libraries using mass spectrometry.

Parallel synthesis techniques aim to prepare collections of single compounds which, once tested, can easily be identified by their sole location in the synthesic array. On the other hand, true combinatorial chemistry produces libraries of compounds as mixtures of variable size which require a deconvolution procedure for identification of the active hits or leads. In the latter case, analytical methods are crucial for the success of the strategy and mass spectrometry plays a major role. If the goal is to identify all the library components, including expected products as well as by-products, various mass spectrometric techniques may be necessary. Library components can be separated according to their mass by increasing mass resolution or by their elution time by coupling liquid chromatography and mass spectrometry. The efficiency of such separation techniques are discussed as a function of the size and the degeneracy of the library. Library members possess common structural features which impart similar fragmentation patterns after ionization in the gas phase. This feature can be exploited by tandem mass spectrometry to specifically detect subfamilies of products. Examples of precursor ion scans, product ion scans and constant neutral loss scans will be shown that facilitate partial characterization of libraries. To solve the difficult problem of the quantitative analysis of libraries, i.e., to evaluate their equimolarity, the use of an evaporative light scattering detector (ELSD) or a chemiluminescent nitrogen detector (CLND) is suggested as more appropriate.

High-performance liquid chromatography coupled with chemiluminescence nitrogen detection for the study of ethoxyquin antioxidant and related organic bases.

The Chemiluminescent Nitrogen Detector (CLND) for use with high-performance liquid chromatography (HPLC) allows for the low-level detection of nitrogen-containing compounds with simple quantitation. The nitrogen selective detector's equimolar response (i.e., equal response for nitrogen independent of its chemical environment) allows for any nitrogen-containing compound to be quantitated as long as the number of nitrogens are known. The HPLC-CLND provides a new detection method for analytes that are not available in large quantities or have unknown chemical or physical characteristics such as oxidation products, metabolites, or impurities. Ethoxyquin is a primary antioxidant that is used to preserve many food products and animal feeds. HPLC-CLND is used in the study of the oxidation products of ethoxyquin because limited quantities of these compounds are available and subsequent calibration curves are difficult to maintain. HPLC-CLND as a new method of detection has been evaluated for its equimolarity of response, linear range, limit of detection, and limit of quantitation.

Apparatus and method for interfacing capillary electrophoresis and chemiluminescence nitrogen detection for the analysis of nitrogen-containing compounds

We have developed an interface that allows the specific detection of nitrogen-containing compounds by using a chemiluminescence nitrogen detector. The feasibility of using this interface was demonstrated by separating and detecting two nitrogen-containing compounds, p-aminosalicylic acid and l-phenylalanine. Although baseline separation was achieved, the theoretical plates were lower when compared to UV detection (25 000 vs. ∼85 000). A sensitivity of 75 ng (∼500 pmol) per injection was achieved with this system which is adequate for pharmaceutical and biotech applications.

Coupling of a Gas-Phase Chemiluminescence Nitrogen Detector and a Capillary Electrophoretic System

A capillary electrophoretic system has been successfully connected to a gas-phase chemiluminescence nitrogen detector via a pneumatic nebulizer interface. The interface, built in-house, consists of a nebulizing gas delivery subsystem, a sheath liquid subsystem, a short spray tube, and a liquid gap. The liquid gap is formed at the point where the spray tube, the separation capillary, and the sheath liquid subsystem meet. The sheath liquid subsystem consists of a grounding connection to complete the electric circuit for the electrophoretic system, a sheath liquid delivery pump, a sheath liquid overflow outlet maintained at atmospheric pressure, and a sheath liquid conduit with a hydrodynamic resistance that is much smaller than that of the separation capillary. The interface operates at the natural self-aspiration rate of the short spray tube. The design ensures that the natural self-aspiration rate of the nebulizer is higher than the maximum electroosmotic flow rate that can be produced in the separation capillary. The flow difference is made up by the sheath liquid which, due to the hydrodynamic resistance differences, is sucked into the liquid gap preferentially from the sheath liquid conduit. Thus, the spray tube and the separation capillary are decoupled from each other hydrodynamically, and any laminar flow-induced additional band broadening in the separation capillary is avoided. Using the combined electrophoretic separation and gas-phase chemiluminescence nitrogen detector system, the mass detection limit for five nucleotide bases used as test compounds was found to be about 10 pmol nitrogen.

Packed-Column Supercritical Fluid Chromatography with Chemiluminescent Nitrogen Detection at High Carbon Dioxide Flow Rates

The use of chemiluminescence nitrogen detection (CLND) coupled with packed-column supercritical fluid chromatography is investigated. The pyrolysis tube design, position of the restrictor, and reaction chamber pressure are shown to affect the response of the detector. By modifying the pyrolysis chamber and controlling the pressure at the reaction chamber, the response of the detector remains constant when different concentrations of methanol modifier are used. This detector design also tolerates high flow rates of decompressed CO 2. As a result, no post-column split is required and the total column effluent is delivered to the CLND. Sensitive detection (limit of detection = 1 ng nitrogen) is achieved at the high flow rates of decompressed CO 2 with 8% methanolmodified CO 2.