Nitrogen-specific Detector Research Articles

A nitrogen-specific detector for high performance liquid chromatography

We describe a nitrogen-specific detector (NSD) for aqueous mobile phase-based high performance liquid chromatography (HPLC). It is based by means of total hydrophilic organic nitrogen detection. Separated analytes are photooxidized online and converted to nitrate, followed by an ultravilet absorbance detector. It features response dependant on the product of nitrogen number in the molecule and its molar concentration, no matter what is ultravilet-absorbing or not. The HPLC equipped with NSD can quantify nitrogen-containing analytes via a sole standard of potassium nitrate for calibration. This results in identical calibration curve for all nitrogen-containing analytes, obviating individual calibration. The limit of detection of NSD is 4.3 μM N/L, and its linear range is up to 4 mM N/L.

Nitrogen Compound Characterization in Fuels by Multidimensional Gas Chromatography.

Certain nitrogen-containing compounds can contribute to fuel instability during storage. Hence, detection and characterization of these compounds is crucial. There are significant challenges to overcome when measuring trace compounds in a complex matrix such as fuels. Background interferences and matrix effects can create limitations to routine analytical instrumentation, such as GC-MS. In order to facilitate specific and quantitative measurements of trace nitrogen compounds in fuels, a nitrogen-specific detector is ideal. In this method, a nitrogen chemiluminescence detector (NCD) is used to detect nitrogen compounds in fuels. NCD utilizes a nitrogen-specific reaction that does not involve the hydrocarbon background. Two-dimensional (GCxGC) gas chromatography is a powerful characterization technique as it provides superior separation capabilities to one-dimensional gas chromatography methods. When GCxGC is paired with a NCD, the problematic nitrogen compounds found in fuels can be extensively characterized without background interference. The method presented in this manuscript details the process for measuring different nitrogen-containing compound classes in fuels with little sample preparation. Overall, this GCxGC-NCD method has been shown to be a valuable tool to enhance the understanding of the chemical composition of nitrogen-containing compounds in fuels and their impact on fuel stability. The % RSD for this method is <5% for intraday and <10% for interday analyses; the LOD is 1.7 ppm and the LOQ is 5.5 ppm.

Unintentional Fatal Intoxications with Mitragynine and O-Desmethyltramadol from the Herbal Blend Krypton

The leaves of Kratom, a medicinal plant in Southeast Asia, have been used as an herbal drug for a long time. At least one of the alkaloids present in Kratom, mitragynine, is a mu-receptor agonist. Both Kratom and an additional preparation called Krypton are available via the internet. It seems to consist of powdered Kratom leaves with another mu-receptor agonist, O-desmethyltramadol, added. O-Desmethyltramadol is an active metabolite of tramadol, a commonly prescribed analgesic. We present nine cases of intoxication, occurring in a period of less than one year, where both mitragynine and O-desmethyltramadol were detected in the postmortem blood samples. Neither tramadol nor N-desmethyltramadol was present in these samples, which implies that the ingested drug was O-desmethyltramadol. The blood concentrations of mitragynine, determined by ultra-performance liquid chromatography-tandem mass spectrometry, ranged from 0.02 to 0.18 μg/g, and O-desmethyltramadol concentrations, determined by gas chromatography with nitrogen-specific detection, ranged from 0.4 to 4.3 μg/g. We believe that the addition of the potent mu-receptor agonist O-desmethyltramadol to powdered leaves from Kratom contributed to the unintentional death of the nine cases presented and conclude that intake of Krypton is not as harmless as it often is described on internet websites.

Nitrogen–phosphorus detection and nitrogen chemiluminescence detection of volatile nitrosamines in water matrices: Optimization and performance comparison

Two nitrogen-specific detection methods, nitrogen–phosphorus detection (NPD) and nitrogen chemiluminescence detection (NCD), were investigated as low cost alternatives to mass spectrometry (MS) with chemical ionization (CI) for analysis of nitrosamines in aqueous samples. NCD showed greater sensitivity to N-nitrosodimethylamine (NDMA) and seven other volatile nitrosamines than did NPD. Instrument detection levels for NDMA were established at 2.6 μg/L and 4.0 μg/L in solvent with 3 μL splitless gas chromatograph (GC) injection for NCD and NPD, respectively. Using a dual-column confirmation method, both NCD and NPD compared favorably with CI-MS results for NDMA analysis in a variety of water sample types. For seven other nitrosamines, both detectors showed excellent accuracy in analyzing high concentrations (greater than 300 ng/L) in complex wastewater matrices, while the accuracy of spike recoveries of very low levels (less than 15 ng/L) in clean matrices varied for each nitrosamine and detection method.

Determination of Imidacloprid in Fruits and Vegetables by Liquid Chromatography with Diode Array and Nitrogen-Specific Chemiluminescence Detection

A national trend is focusing on expanding usage of environmentally friendly nitrogenous pesticides and herbicides. However, some of these chemicals are undetectable by any gas chromatographic method due to their thermal lability or nonvolatility. Using imidacloprid as a model compound, this laboratory has developed a simple, effective, and reliable liquid chromatography (LC) method to address the inherent difficulties of these chemicals. Focusing on nitro, nitrate, and/or nitrite chromophore groups and components contained in the chemical structure of most nitrogenous compounds, LC with diode array detector and a nitrogen-specific detector in tandem enabled determination and confirmation analyses in series. Furthermore, using 2 mixtures of solvents in conjunction with 2-stage solid-phase extraction cleanup on aminopropyl/Florisil cartridges to eliminate and/or minimize interferences resulting from free amino acids, peptides, proteins, pigments, and lipids in the plant tissues made the qualitative and quantitative analysis simple, fast, and cost effective. This method demonstrated the potential for becoming a viable analysis tool for many other types of nonvolatile nitrogenous chemicals.

Highly sensitive determination of a polymeric hindered amine light stabilizer in polypropylene by reactive thermal desorption–gas chromatography using nitrogen-specific detection

Highly sensitive and specific determination of trace amounts of a polymeric hindered amine light stabilizer (HALS) in polypropylene (PP) materials could be established by improving reactive thermal desorption–gas chromatography (RTD–GC) in the presence of an organic alkali, tetramethylammonium hydroxide. By using nitrogen–phosphorus detection, highly selective detection of the HALS-related components was attained. In addition, the use of a polar poly(ethylene glycol) separation column alleviated the adsorption of minor specific pyrolysis products. This modified RTD–GC method allowed the determination of the polymeric HALS ( M r 1900) in PP even for trace concentrations between 100 and 500 ppm, through observing selectively the characteristic products containing a tetramethylpiperidine moiety, which had been impossible to detect under the previous RTD–GC conditions using a non-polar separation column and conventional flame ionization detection.

Determination of remifentanil in human blood by capillary gas chromatography with nitrogen-selective detection

A validated method for the determination of remifentanil in human blood, applicable to all therapeutic concentrations, using capillary GC with nitrogen-specific detection and fentanyl as the internal standard has been developed. Citrated whole blood samples were extracted into 1-chlorobutane following precipitation of proteins with methanol. The drugs were back extracted into 10 m M HCl and re-extracted into methanol–1-chlorobutane. The extracts were reconstituted in methanol and injected onto a 25-m BPX-5 column. The lower limit of quantitation was 0.2 ng/ml with within- and between-day coefficients of variation of less than 15%.

Determination of zwitterionic and cationic surfactants by high-performance liquid chromatography with chemiluminescenscent nitrogen detection

The use of chemiluminescent nitrogen specific detection (CLND) combined with an HPLC separation allows for the identification and quantification of cationic and zwitterionic surfactants. The CLND provides equimolar responses, based on the amount of nitrogen within any compound. This allows for the detection of any nitrogen containing surfactant. Reversed-phase separation methods using cyano columns are developed for cationic and zwitterionic (sulfobetaine) surfactant mixtures. The limits of detection for these surfactants are in the single micromolar range (1 ng N). A linear response was obtained ( R 2=0.9981) between 50 μ M and 5 m M. The methodology was then applied to the determination of an industrial zwitterionic surfactant, Rewoteric AM CAS U [coco(amidopropyl)hydroxyldimethylsulfobetaine].

Determination of relative UV response factors for HPLC by use of a chemiluminescent nitrogen-specific detector

Ultraviolet (UV) absorbance is the most widely used detection method for high-performance liquid chromatography (HPLC) separations. In pharmaceutical analysis, purity determinations often include quantitation of related impurities based on relative HPLC peak areas obtained at a specific wavelength. In order for this quantitation to accurately reflect weight percentages of impurities, the relative UV response factors (absorptivities) at the given wavelength must be known. In this work, we present a convenient method for determining relative UV response factors on-line, without isolation or purification of impurities, without standards, and without requiring known analyte concentrations. The procedure described makes use of a chemiluminescent nitrogen-specific HPLC detector (CLND) in conjunction with a UV detector. The CLND response is directly proportional to the number of moles of nitrogen in each eluting peak, and can, therefore, be used to determine relative amounts of each nitrogen-containing impurity present in the sample, provided the molecular formulas are known (e.g. from exact mass LC–MS). It is a simple matter, then, to determine the relative UV response factors from the UV area ratios obtained for the same sample. The feasibility and accuracy of this method is demonstrated for gradient HPLC separations of commercially available compounds of widely varying structures. Finally, the method's utility in obtaining accurate mass balance is demonstrated by application to photodegradation of nifedipine.

Developmental renal tubular toxicity: a hypothetical framework and experimental approaches

Background: The antineoplastic agent ifosfamide is capable of causing renal damage in children. It is not known why children younger than 3 years experience more nephrotoxicity, regardless of the concurrent administration of the synthetic thiol compound sodium 2-mercaptoethane sulfonate (mesna). The ability of mesna to form stable nontoxic thioether compounds occurs at the expense of intracellular glutathione, which when decreased may predispose kidney cells to localized oxidative stress, resulting in renal damage. We present a hypothetical framework that assumes that the renal tubular cell is capable of producing nephrotoxic metabolites, and that this happens more often among young children, either because of higher cytochrome P450 (CYP) activity or lower levels of antioxidants. Objective: The aim of this study was to show whether the prodrug ifosfamide is cytotoxic to renal proximal tubule cells once they are depleted of glutathione. Methods: Human kidney microsomes and CYP 3A4, 3A5, and 3A7 Supersomes™ were incubated with 1 mM ifosfamide and the results analyzed by means of gas chromatography using thermionic nitrogen-specific detection (GC/TSD). Microsomes were separated by means of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis on 10% gels, transferred to nitrocellulose, and immunoblotted with CYP 3A4 and 3A5 antigen. Finally, porcine renal proximal tubular cell monolayer (LLC-PK-1) was used to determine whether ifosfamide is cytotoxic to renal tubule cells once the cells have been depleted of glutathione with 250 μM buthionine sulfoxime (BSO). Results: CYP 3A4 and 3A5 were found to be present in human kidney microsomes. The ability of kidney microsomes to metabolize ifosfamide to its 2-dechloroethylifosfamide and 3-dechloroethylifosfamide metabolites was confirmed. Furthermore, pretreatment of LLC-PK-1 cells for 24 hours with 250 μM of the glutathione-depleting substance BSO led to a significant decrease in the number of viable cells when cells were incubated with either 100 μM or 1 mM ifosfamide for either 72 or 96 hours. BSO pretreatment for 24 hours followed by ifosfamide treatment for 24 or 48 hours did not significantly decrease the number of viable LLC-PK-1 cells. Conclusions: The human kidney possesses the necessary enzymatic machinery (CYP 3A4 and 3A5) to metabolize ifosfamide to nephrotoxic metabolites. Decreasing the intracellular levels of glutathione in renal proximal tubule cells subjects them to damage that results in a decrease in the number of viable cells. Additional studies on the ontogeny of these processes are in progress.

Application and validation of a urinary methadone metabolite (EDDP) immunoassay to monitor methadone compliance.

A total of 1381 urine specimens were screened using a Microgenics CEDIA urinary primary methadone metabolite (2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine; EDDP) immunoassay (cut-off calibrator concentration of 100 microg/L) in combination with a Dade Behring EMIT urinary methadone immunoassay (cut-off calibrator concentration of 300 microg/L). Of these, 642 (46%) were found to be positive using the EDDP assay but only 541 (39%) were found to be positive by the methadone assay. Out of the 108 specimens which were EDDP-positive but negative by the methadone assay, 47 (7%) could not be confirmed as positive using the routine in-house method of gas chromatography incorporating nitrogen-specific detection. Of these 47 results, 38 were re-screened using a more sensitive gas chromatography-mass spectrometry (GC-MS) technique, which demonstrated the presence of EDDP in every case. There was insufficient specimen to analyse the remaining nine samples. There were seven specimens which gave negative results by the EDDP and GC-MS assays but which had methadone concentrations ranging from 4000 to 37,500 microg/L. These were therefore presumed to be 'spiked' with methadone, i.e. to have had methadone added to the specimen to yield positive screening results and simulate compliance. It is concluded that the Microgenics CEDIA EDDP assay is a sensitive and reliable technique to determine the compliance of subjects prescribed methadone for opiate detoxification and maintenance.

Using amphetamine isomer ratios to determine the compliance of amphetamine abusers prescribed dexedrine.

The amphetamine isomer ratios (l-amphetamine/d-amphetamine) in 373 urine specimens submitted for analysis over a two-year period have been determined using a chiral derivatizing agent in conjunction with a gas chromatograph fitted with a nitrogen-specific detector. All of the specimens were collected from known or suspected amphetamine abusers, some of which were prescribed dexedrine for maintenance and detoxification. The mean (+/- 1 standard deviation [SD]) l/d-amphetamine isomer ratio for 147 specimens from compliant subjects prescribed dexedrine was 15.0% (+/- 4.9%). The mean (+/- 1 SD) l/d-amphetamine isomer ratio from 165 subjects abusing illicit amphetamine was 98.5 (+/- 27.5%). The calculation of l/d-amphetamine isomer ratios in urine has been found to be a rapid method for determining the compliance of subjects prescribed dexedrine and is therefore a useful technique for the continued management of amphetamine abusers. In addition, 17 specimens of illicit amphetamine powder (assumed to be a racemic mixture) were submitted to the laboratory for analysis. Using a combination of gas chromatography with and without chiral derivatization, the powders were found to have a mean l/d-amphetamine isomer ratio of 89.2% (range 72.2% to 98.3%) and mean purity (w/w) of 21.5% (range 3.4% to 71.0%) relative to pure dl-amphetamine substance.

Investigation of the boiled potato flavor by human sensory and instrumental methods

The flavor of boiled potatoes was investigated using sensory and instrumental methods to get basic information concerning sensory features and aroma compounds for use in breeding research and quality control. The sensory odor profiles of three genotypes were found to differ especially in negative sensations like “burnt”, “fodder” and “musty”. The aroma volatiles were isolated by a modified simultaneous steam distillation and extraction method. Nineteen character impact compounds of boiled potato aroma were pointed out by gas chromatography with nitrogen specific detection, gas chromatography —mass spectrometry and gas chromatography —olfactometry. The comparison of the quantified odor impressions in three genotypes by chromatography —olfactometry revealed the substances responsible for the typical boiled potato odorants and off-flavor notes.

Assessing Compositional Changes of Nitrogen Compounds during Hydrotreating of Typical Diesel Range Gas Oils Using a Novel Preconcentration Technique Coupled with Gas Chromatography and Atomic Emission Detection

This paper describes the identification of nitrogen-containing compounds in a typical feed for diesel oil hydrotreating and how their individual concentrations change upon hydrotreating over a conventional sulfided CoMo/Al2O3 catalyst at commercial conditions. A preconcentration procedure followed by gas chromatographic (GC) analysis utilizing a highly sensitive nitrogen-specific detector (atomic emission detector) allowed the quantitative analysis of individual nitrogen-containing compounds (N compounds) at levels as low as 0.05 μg N/mL. The nitrogen compounds in the feed and products were identified by comparison with reference compounds as well as by high-resolution GC/mass spectrometric characterization. The relative reactivities of individual compounds in the diesel fuel feed were determined and the most refractory compounds identified. Alkyl-substituted carbazoles were found to be the major compound class in the feed and to be among the least reactive N compounds in the feed. Just as in the case of ...

Overview of stobadine bioanalysis: evaluation and application in pharmacokinetics.

Besides its many pharmacodynamic actions, the pyridoindole stobadine was found to exert antioxidant activity and thus possesses the potential to protect various tissues against oxidative stress. This overview is focussed on both the evaluation of the chemical procedures used in the bioassay of stobadine and its metabolites and on the comparison of their quality in the light of applicability for preclinical and clinical pharmacokinetic experiments. All methods and applications were performed at the Institute of Experimental Pharmacology, SASc in Bratislava, Slovakia. In pharmacokinetic and toxicokinetic studies, [3H]-labeled stobadine dihydrochloride was administered intravenously or orally to rats in single and repeated doses. Liquid-liquid extraction was used for selective isolation of stobadine and its metabolites from biological matrix, followed by liquid scintillation quantification. A TLRC method was developed both to check the radiochemical purity of [3H]-stobadine and to quantify the labeled drug in rat plasma. A spectrofluorometric approach was used for determination of stobadine in dog serum and urine after its administration in the form of either the dihydrochloride or the dipalmitate. The method allowed us to perform a bioavailability study and a long-term toxicological study. The HPLC method with a limit of detection of 10 ng/ml of plasma proved suitable for calculating the compartmental pharmacokinetic parameters of both salt forms of stobadine administered to dog and man. This method was based on solid-phase extraction procedure by using Separcol SI C18 cartridges. In a GC method, the combination of capillary column separation and nitrogen-specific detection permitted the assay of serum stobadine concentrations as low as 5 ng/ml. The detection limit of the GC/MS method was 1 ng/ml of plasma or of phosphate buffer saline. This method was used for a bioequivalence study of two stobadine dipalmitate dosage forms and for a transdermal penetration study of stobadine acyl derivatives. All the developed assays proved to be appropriate for low-concentration determination of stobadine in a wide range of pharmacokinetic studies.

Isotope dilution GC-MS of benzylpenicillin residues in bovine muscle.

The German provisional prevalidated official method for the determination of residues of benzylpenicillin in muscle (beef) which uses gas chromatography with nitrogen-specific detection (GC-NPD) was successfully adapted to gas chromatography-mass spectrometry (GC-MS) with selected-ion monitoring. The programmed temperature vaporisation injection technique used in the original method was substituted by on-column injection. An isotope dilution procedure for benzylpenicillin using 13C2-labelled benzylpenicillin as internal standard was developed to improve the reliability of the GC-MS results. The spectral overlap of this labelled standard and the analyte caused a non-linear relationship in calibration for which linear bracketing and single-point calibration were compared. Single-point calibration proved to be the superior method, showing less deviation from the 'true' value, while at the same time requiring fewer calibration measurements. A comparison of the results obtained by using (i) external standardisation only, (ii) a close analogue of the analyte (phenoxymethylpenicillin) and (iii) 13C2-labelled Pen G as internal standard showed, as was to be expected, that varying recoveries are best corrected by using the isotope-labelled standard.

Specific gas chromatographic analysis of diethylcarbamazine in human plasma using solid-phase extraction

Diethylcarbamazine (DEC, 1-diethylcarbamyl-4-methylpiperazine) is an antiparasitic piperazine derivative used in the treatment of lymphatic filariasis. DEC-N-oxide is a major metabolite in humans which has antifilarial activity. Gas chromatographic analysis of DEC in plasma can be complicated by the presence of the metabolite, since the thermally unstable DEC-N-oxide is converted to a material which coelutes with DEC under the conditions of the analysis. We now report a method to separate DEC-N-oxide from DEC in plasma using solid-phase extraction with subsequent gas chromatographic analysis using a nitrogen specific detector. 1-Diethylcarbamyl-4-ethylpiperazine (E-DEC) was the internal standard. The standard curve of DEC is linear in the range of 10 to 200 ng/ml. The limit of detection is 4 ng/ml. Reproducibility at 10, 100 and 200 ng/ml concentration points of the standard curve gives coefficients of variation of 6.1%, 7.8% and 1.6%, respectively. Recovery following solid-phase extraction is 99.3% for DEC and 94.8% for the internal standard. This sensitive and specific analytical method is suitable for pharmacokinetic studies of DEC.

Speciation of Nitrogen Compounds in Gasoline and Diesel Range Process Streams by Capillary Column Gas Chromatography with Chemiluminescence Detection

This paper describes the development of a gas chromatographic method that uses nitrogen-specific detection based on chemiluminescence. A highly sensitive nitrogen-specific detector (Antek 705D) coupled to a Hewlett-Packard gas chromatograph is optimized to speciate nitrogen compounds in gasoline and diesel range process streams. Under optimized conditions, the nitrogen-to-carbon selectivity is greater than 106. The nitrogen chemiluminescence detector (NCD) provides a uniform response to different classes of nitrogen compounds and can detect concentration of individual components down to 100 ppb nitrogen. The detector's linear response in a range of 0.2 to 54 ppm nitrogen for an individual nitrogen component is established using several aliphatic and aromatic nitrogen-containing compounds. Gasoline and diesel range streams containing any level of total nitrogen can be analyzed after appropriate dilution so that each nitrogen component is below 50 ppm. In addition to the nitrogen speciation, total nitrogen can simultaneously be determined with reasonable accuracy using the NCD system. The repeatability and accuracy of the nitrogen quantitation is found to be within 7% at the 95% confidence level based on six to seven measurements.

Pharmacokinetics of intravenous and oral prethcamide in horses

The respiratory stimulant prethcamide is a mixture of equal parts of crotethamide and cropropamide. A specific and sensitive gas chromatographic method for the determination of crotethamide and cropropamide in horse plasma and urine is described. Both components of prethcamide were extracted from plasma and urine into dichloromethane. The extracts were analyzed by capillary gas chromatography with thermionic detection in the nitrogen-specific detection mode. The lower limits of quantitation were 4.0 ng ml −1 of plasma and 10.0 ng ml −1 of urine. Calibration curves were linear from 2.0–100 ng ml −1 of plasma for both components. Pharmacokinetic parameters for crotethamide and cropropamide after intravenous and oral dosing were estimated by analysis of plasma concentration versus time data. The total plasma clearance of cropropamide was greater than that of crotethamide and both values were greater than 5 ml min −1 kg −1. Renal clearance values of the two drugs were comparable and were much less than estimates of filtration clearance values in horses, indicating extensive re-absorption of both components from the renal tubules. Both compounds were metabolized by N-demethylation of the [(dimethylamino)-carbonyl]-propyl moiety and these metabolites were excreted in urine. The method was demonstrated to be suitable for detecting illicit administration of prethcamide to competition horses.

Analysis of diethylcarbamazine and diethylcarbamazine-N-oxide by gas chromatography

Diethylcarbamazine (DEC, 1-diethylcarbamyl-4-methylpiperazine) is an antiparasitic piperazine derivative used in the treatment of lymphatic filariasis caused byWuchereria bancrofti, Brugia malayi orBrugia timori. DEC-N-oxide is a major metabolite in humans and has antifilarial activity. In carrying out pharmacokinetic studies, gas chromatographic analysis of DEC in plasma can be complicated by the presence of the metabolite, since the thermally unstable DEC-N-oxide is converted back to a material which coelutes with DEC under the conditions of the analysis. We now report a method to separate DEC-N-oxide from DEC in plasma using solid phase extraction with subsequent gas chromatographic analysis using a nitrogen specific detector. One-diethylcarbamyl-4-ethylpiperazine (E-DEC) was the internal standard. The standard curve of DEC was linear in the range of 10 to 200 ng/ml as described by Y=0.0350+0.0128X, R2=0.999. The limit of quantitation was 4 ng/mL. Reproducibility at 10, 100 and 200 ng/mL concentration points of the standard curve gave coefficient variations of 6.1%, 7.8% and 1.6%, respectively. The recovery following solid phase extraction was 99.3% for DEC and 94.8% for the internal standard. This sensitive and specific analytical method is suitable for pharmacokinetic studies of DEC.