Determination of 10 alpha-methoxy-9,10-dihydrolysergol, a nicergoline metabolite, in human urine by high performance liquid chromatography.

A specific method for the determination of 10 alpha-methoxy-9,10-dihydrolysergol, a nicergoline metabolite (metabolite 2), in urine is described. Metabolite 2 was well separated from the urine components on a reversed phase column, Hypersil ODS 5 microns, using an acetonitrile:pH 3.5 phosphate buffer (40:60, v/v) as the mobile phase at a flow rate of 1 mL/min. UV detection was set up at 220 nm. After addition of a known amount of lysergamide as the internal standard, the compounds were extracted from alkalysed urine on a pre-packed glass column (Extrelut 1) with dichloromethane. With 0.5 mL urine, concentrations down to 0.56 mumol/L could be determined.     

新型薄层色谱内标法测定葛根素含量

以大豆甙元为内标物在高效硅胶薄层板上采用新型内标法测定了葛根素注射液、中药葛根及中成药玉泉丸中葛根素的含量,建立了新型TLC内标法测定葛根素的新方法。结果表明：用大豆甙元作为内标物测定葛根素可以满足TLC内标物的一般条件,新型TLC内标法测定葛根素的含量具有测定结果准确,精密度好,回收率高等优点。     

Chiral determination of various adrenergic drugs by thin-layer chromatography using molecularly imprinted chiral stationary phases prepared with alpha-agonists

Thin-layer chromatography (TLC) based on molecularly imprinted polymers (MIPs) of alpha-agonists as chiral stationary phases was applied to the determination of enantiomers of various adrenergic drugs including alpha- and beta-agonists and beta-antagonists (beta-blockers). In this study, three MIPs imprinted with (+)-ephedrine, (+)-pseudoephedrine and (+)-norephedrine plus a non-imprinted polymer (non-MIP) were prepared, processed and coated on a glass support as thin layers. then enantiomeric determination of adrenergic drugs was carried out by development of their racemates on the TLC plates, using established conditions. From the results, the racemates of the compounds used as print molecules were well separated into two isomers on the MIP-plates, except on the plate based on MIP of (+)-norephedrine. Most adrenergic drugs structurally related to print molecules were completely resolved into two spots with the MIP plates. In general the retention of (+)-isomers (or 1S-isomers) was greater than that of (-)-isomers (or 1R-isomers), indicating the stereoselectivity of the MIPs with the former isomers. Moreover, the role between the chemical structures of the analytes with chiral recognition of the MIPs has been investigated. The proposed method enables rapid determination of enantiomers and screening of large numbers for optical purity of adrenergic drugs.     

Rapid,simple, and highly sensitive analysis of drugs in biological samples using thin-layer chromatography coupled with matrix-assisted laser desorption/ionization mass spectrometry

Rapid and precise identification of toxic substances is necessary for urgent diagnosis and treatment of poisoning cases and for establishing the cause of death in postmortem examinations. However, identification of compounds in biological samples using gas chromatography and liquid chromatography coupled with mass spectrometry entails time-consuming and labor-intensive sample preparations. In this study, we examined a simple preparation and highly sensitive analysis of drugs in biological samples such as urine, plasma, and organs using thin-layer chromatography coupled with matrix-assisted laser desorption/ionization mass spectrometry (TLC/MALDI/MS). When the urine containing 3,4-methylenedioxymethamphetamine (MDMA) without sample dilution was spotted on a thin-layer chromatography (TLC) plate and was analyzed by TLC/MALDI/MS, the detection limit of the MDMA spot was 0.05 ng/spot. The value was the same as that in aqueous solution spotted on a stainless steel plate. All the 11 psychotropic compounds tested (MDMA, 4-hydroxy-3-methoxymethamphetamine, 3,4-methylenedioxyamphetamine, methamphetamine, p-hydroxymethamphetamine, amphetamine, ketamine, caffeine, chlorpromazine, triazolam, and morphine) on a TLC plate were detected at levels of 0.05 − 5 ng, and the type (layer thickness and fluorescence) of TLC plate did not affect detection sensitivity. In addition, when rat liver homogenate obtained after MDMA administration (10 mg/kg) was spotted on a TLC plate, MDMA and its main metabolites were identified using TLC/MALDI/MS, and the spots on a TLC plate were visualized by MALDI/imaging MS. The total analytical time from spotting of intact biological samples to the output of analytical results was within 30 min. TLC/MALDI/MS enabled rapid, simple, and highly sensitive analysis of drugs from intact biological samples and crude extracts. Accordingly, this method could be applied to rapid drug screening and precise identification of toxic substances in poisoning cases and postmortem examinations.     

Separation and quantitation of nicergoline and related substances by high-performance liquid chromatography/atmospheric pressure ionization mass spectrometry

Summary This study demonstrated the utility of high-performance liquid chromatography/atmospheric pressure ionization mass spectrometry (HPLC/API-MS) in the investigation of 10-methoxy-1,6-dimethylergoline-8-methanol 5-bromonicotinic acid ester (Nicergoline) and its related substances. The analysis was performed by using an ODS column with ammonium acetate and methanol mixture as the mobile phase. Nicergoline and its related compounds could be characterized by HPLC/API-MS in terms of their molecular weight. The use of multiple ion detection techniques for the quantitation of these compounds was also investigated. The detection limits of nicergoline and its related substances were 5 to 10 ng each at a signal-to-noise ratio of 4. The method was also applied to the study of the decomposition products of nicergoline in simulated gastric and intestinal fluids.     

Hydroxylation of the silica in microfabricated thin layer chromatography plates as probed by time‐of‐flight secondary ion mass spectrometry and diffuse reflectance infrared Fourier transform spectroscopy

Microfabricated silica thin layer chromatography (TLC) plates have previously been prepared on patterned carbon nanotube forests. The high temperatures used in their fabrication reduce the number of hydroxyl groups on their surfaces. Fortunately, silica can be rehydroxylated. In diffuse reflectance infrared Fourier transform spectroscopy (DRIFT), a silanol peak below 3740 cm^?1 indicates a well‐hydroxylated silica surface that is fit for chromatography. Hydroxylations of our materials with HF are so effective that it is not possible to discern the position of this peak. In contrast, this signal is discernable when the plates are treated with NH₄OH. To find a more convenient method for studying the surfaces of TLC plates, time‐of‐flight secondary ion mass spectroscopy (ToF‐SIMS) was considered. ToF‐SIMS is advantageous because multiple microfabricated TLC plates must be scraped to obtain enough silica for one DRIFT analysis, while static SIMS can be performed on very small regions (500 × 500 µm² or less) of individual plates. Ratios of the SiOH⁺ and Si⁺ ToF‐SIMS signals for microfabricated TLC plates correlated well with ~3740 cm^?1 silanol peaks from DRIFT. Thus, SIMS allows direct analysis of all of our treated and untreated plates, including those hydroxylated with HF. The best hydroxylation condition for HF, which was better than any studied for NH₄OH, was around 150 ppm at room temperature. The best hydroxylation condition for NH₄OH was 50 °C for 72 h. ToF‐SIMS versus DRIFT results of commercial TLC plates were also obtained and evaluated. Copyright © 2014 John Wiley & Sons, Ltd.     

A fast high throughput method for the determination of acidity constants by capillary electrophoresis. 3. Basic internal standards

A set of 25 monoprotic bases is proposed as internal standards for pK(a) determination by capillary electrophoresis. The pK(a) of the bases is determined and compared with available literature data. The capillary electrophoresis internal standard method offers numerous advantages over other typical methods for pK(a) determination, especially of analysis time and buffer preparation. However, it requires disposing of appropriate standards with reference pK(a) value. The set of bases established in this work together with the set of acids previously established provide a reference set of compounds with well-determined acidity constants that facilitate the process of selecting appropriate internal standards for fast pK(a) determination by capillary electrophoresis in high throughput screening of pharmaceutical drugs. In addition, the performance of the method when acidic internal standards are used for the determination of acidity constants of basic internal standards has also been tested. Although higher errors may be expected in this case, good agreement is observed between determined and literature values. These results indicate that in most cases structural similarity between the analyte and the internal standard might not be an essential requirement in the internal standard method.     

Simultaneous determination of methylxanthines in different types of tea by a newly developed and validated TLC method

The aim of this paper is to develop a new simple, fast and economical method for simultaneous quantitative determination of methylxanthine compounds based on TLC combined with image analysis. To obtain certain results, both extraction and chromatographic separation were optimized. The optimum extraction conditions were maceration in ethanol-water 8:2, v/v. The chromatographic separations were done on the silica gel F(254) TLC plates developed with chloroform-dichloromethane-isopropanol, 4:2:1 v/v/v. Detection was performed under UV lamp at 254?nm and the evaluation of the chromatographic plate was based on digital processing of chromatographic images. The developed TLC method was validated for parameters such as specificity, linearity and range, LOD and LOQ, precision, robustness and accuracy. This method was then applied for determination of caffeine, theobromine and theophylline in different types of tea, commercially available. Moreover, the content of methylxanthines detected and determined in commercial tea samples can be used as chemical marker in quality control.     

Application of molecular-secondary-ion mass spectrometry for drug metabolism studies. I. Direct analysis of conjugates by thin-layer chromatography-secondary-ion mass spectrometry

Molecular-secondary-ion mass spectrometry (SIMS) is a suitable method for the analysis of nonvolatile substances such as conjugated metabolites of drugs. We have developed a simple method for the direct SIMS measurement of conjugates following thin-layer chromatography without any extraction procedure. After separation with a butanol-acetic acid-ethanol-water (3:1:1:1, v/v) system, the spot was cut out and attached to a SIMS probe. The conjugates of p-nitrophenol and 4-hydroxyantipyrine were measured. The quantitative application of the method is also discussed, using deuterium-labelled internal standards for p-nitrophenol conjugates.     

Stability indicating methods for the determination of some fluoroquinolones in the presence of their decarboxylated degrades

Two stability-indicating methods, namely densitometric TLC and derivative spectrophotometry for the determination of the fluoroquinolone antibacterials lomefloxacin (Lfx), moxifloxacin (Mfx), and sparfloxacin (Sfx) in the presence of their acid degrades are described. Acid degradation was adopted and the main decarboxylated product separated by TLC. Degradation products were identified confirming a previously mentioned degradation scheme. The densitometric method is based on the separation of the intact drug from its acid degradation product on silica gel G plates using different mobile phases and the spots of the intact drugs were scanned at 288, 290, and 292 nm for Lfx, Mfx, and Sfx, respectively. The derivative spectrophotometric method utilizes first derivative D(1) UV spectrophotometry with zero crossing points at 295.2 nm for Lfx, 280.4 and 303.4 nm for Mfx, and 280.8 nm for Sfx. Regression analysis of Beer's plots showed good correlation in the concentration ranges 0.2-1.2, 0.1-1.4, and 0.5-2.0 microg/spot for Lfx, Mfx, and Sfx, respectively, in the densitometric method and 2-16 microg/ml for all drugs in the derivative spectrophotometric method. The proposed methods were successfully applied for the determination of the investigated drugs in bulk powder with mean percentage accuracy ranges from 98.93 to 101.25% for the TLC method and from 98.18 to 100.35% for the D(1) method. The proposed methods were also applied for the determination of the investigated drugs in their pharmaceutical dosage forms and their validity was assessed using the standard addition technique with mean percentage recovery ranging from 100.25 to 101.70% in the TLC method and from 99.27 to 102.12% in the D(1) method. The selectivity of the proposed methods was tested by the analysis of laboratory-prepared mixtures containing different percentages of the studied drugs and their acid degrades. The proposed methods were found selective for the determination of the intact drugs in the presence of up to 90% of their degrades in the TLC method and 70% for Lfx and 90% for Mfx and Sfx in the D(1) method.     

Comparison of ICP-MS and SIMS techniques for determining uranium isotope ratios in individual particles

The determination of uranium isotope ratios in individual particles is of great importance for nuclear safeguards. In the present study, an analytical technique by inductively coupled plasma mass spectrometry (ICP-MS) with a desolvation sample introduction system was applied to isotope ratio analysis of individual uranium particles. In ICP-MS analysis of individual uranium particles with diameters ranging from 0.6 to 4.2 μm in a standard reference material (NBL CRM U050), the use of the desolvation system for sample introduction improved the precision of ²³⁴U/²³⁸U and ²³⁶U/²³⁸U isotope ratios. The performance of ICP-MS with desolvation was compared with that of a conventionally used method, i.e., secondary ion mass spectrometry (SIMS). The analysis of test swipe samples taken at nuclear facilities implied that the performance of ICP-MS with desolvation was superior to that of SIMS in a viewpoint of accuracy, because the problems of agglomeration of uranium particles and molecular ion interferences by other elements could be avoided. These results indicated that ICP-MS with desolvation has an enough ability to become an effective tool for nuclear safeguards.     

Determination of antimigraine compounds rizatriptan, zolmitriptan, naratriptan and sumatriptan in human serum by liquid chromatography/electrospray tandem mass spectrometry

Development of a rapid, sensitive and selective method for the determination of antimigraine drugs from human serum is essential for understanding the pharmacokinetics of these drugs when administered concurrently. Solid phase extraction (SPE) using Oasis HLB was used to extract the drugs (sumatriptan, naratriptan, zolmitriptan and rizatriptan) and the internal standard bufotenine from serum. A method based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed and validated to simultaneously quantitate these antimigraine drugs from human serum. The precursor and major product ions of the analytes were monitored on a triple quadrupole mass spectrometer with positive ion electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode. The base peak in all the analytes is formed by alpha cleavage associated with protonation of the secondary amine. Mechanisms for the formation of the collision-induced dissociation products of these antimigraine compounds are proposed. Linear calibration curves were generated from 1-100 ng/mL with all coefficients of determination greater than 0.99. The inter- and intraday precision (%RSD) were less than 9.3% and accuracy (%error) was less than 9.8% for all components. The limits of detection (LOD) for the method were 250 pg/mL for sumatriptan and 100 pg/mL for the remaining analytes based on a signal-to-noise ratio of 3.     

Enantioselective drug monitoring of (R)- and (S)- propranolol in human plasma via derivatization with optically active (R,R)-O,O-diacetyl tartaric acid anhydride

A sensitive high-performance liquid chromatographic method was developed for the stereoselective assay of (R)- and (S)-propranolol in human plasma. The method involves diethyl ether extraction of the drugs and a racemic internal standard, N-tert.-butylpropranolol, followed by derivatization of the compounds with the chiral reagent (R,R)-O,O-diacetyl tartaric acid anhydride. The resulting diastereomeric derivatives were separated isocratically on a reversed-phase column. Quantitation was achieved by the peak-height ratio method with reference to the internal standard. The assay was accurate and reproducible in the concentration range 1-100 ng of (R)- and (S)-propranolol per ml plasma, using fluorescence detection at lambda ex 290 nm and lambda em 335 nm. The applicability of this method was demonstrated for the determination of concentration-time profiles of propranolol enantiomers in the course of comparative pharmacokinetic studies.     

Development of a reversed-phase thin-layer chromatographic method for artemisinin and its derivatives

In this study a clear separation between seven analogues of artemisinin on thin-layer chromatography (TLC) is presented. The developed TLC method is carried out on a RP-C18 thin-layer plate using acetonitrile-water (50:25 v/v) as the mobile phase. Spots are visualized by derivatization with an acidified 4-methoxybenzaldehyde reagent in methanol-water. This method allows the separation of a diverse group of compounds that have versatile hydrophilic/lipophilic characteristics; namely artemisinin, artesunate (AS), artelinic acid (AL), arteether (AE), both isomers of artemether (AM) (alpha and beta), dihydroartemisinin, and desoxyartemisinin. Separation of some degradation products and impurities, down to 2%, allows quality control and stability investigation of all actives in raw material and pharmaceutical formulations. The method is further developed via densitometric measurement for quantitative determination purposes for AL and AS. The derivatization technique is evaluated, showing good stability and reproducibility of the coloring process. Percent relative standard deviation values are less than 5% for replicates, and linearity is obtained in the range of 0.5 to 8 microg. A comparative study with high-performance liquid chromatography (HPLC) on a C18 column, applying the same mobile phase, proves the suitability of the TLC method, in which almost all presented analytes are separated from each other. In contrast, HPLC requires at least a 20-min analysis to chromatograph all of the compounds and only betaAM and AE are clearly separated from each other and from the other compounds.     

Selective separation and determination of isoproterenol on thin layers of bismuth silicate ion-exchanger

A simple and sensitive method for the separation and determination of isoproterenol from other doping drugs has been developed on thin layers of bismuth silicate, a synthetic inorganic ion exchanger as adsorbent in thin layer chromatography (TLC). A mixture of methanol and 0.1 mol/L formic acid (3:7, v/v) was employed as the mobile phase. The development time was 32 min. The quantitative measurement were performed with a Camag TLC Scanner-3 at wavelength (λ) of 410 nm. The isoproterenol recovery in this procedure was 98.9%. The linear correlation coefficient was greater than 0.9871 and the relative standard deviation (RSD) was less than 0.94. The limit of detection (LOD) and limit of quantification (LOQ) were 7.7×10^-7mol/L and 3.85 ×10^-6mol/L, respectively. This method has been applied in the determination of isoproterenol in dosage forms and in biological fluids.     

High performance liquid chromatography with UV detection for the simultaneous determination of sympathomimetic amines using 4-(4,5-diphenyl-1H-imidazole-2-yl)benzoyl chloride as a label

A high performance liquid chromatographic method has been developed for the simultaneous determination of (+/-) fenfluramine (Fen) and phentermine (Phen) in addition to three other sympathomimetic amines-ephedrine (E), norephedrine (NE) and 2-phenylethylamine (2-PEA), using cyclohexylamine (CX) as an internal standard in plasma. The compounds were derivatized with 4-(4,5-diphenyl-1H-imidazole-2-yl)benzoyl chloride (DIB-Cl) to give the DIB-derivatives. The derivatives were then separated using an isocratic HPLC system with UV detection. The limits of detection for Fen, Phen, E, NE and 2-PEA in plasma ranged from 0.32 to 22.9 pmol on column at a signal-to-noise ratio of 3. The recoveries following alkaline extraction from plasma samples of known concentrations were found to be more than 94% for the studied compounds. This method might be useful for the screening of the studied sympathomimetic amines in human plasma samples in forensic as well as toxicological studies. Furthermore, the developed method was modified for the simultaneous determination of Fen and Phen in human and rat plasma using fluoxetine as an internal standard. The methods are reproducible and precise. Finally, the two drugs were administered intraperitoneally to rats in combination, and their plasma levels over the investigated time course were successfully determined.     

Ion-pair extraction and high-performance liquid chromatographic determination of tianeptine and its metabolites in human plasma, urine and tissues

An isocratic reversed-phase ion-pair liquid chromatographic method for the determination of tianeptine and its two main metabolites in plasma, urine and tissues, using an internal standard, is reported. The influence of two stationary phases on the retention of the drugs was studied. The drugs were extracted as ion pairs, using a heptane-octanol-tetraheptylammonium bromide mixture (98:2:0.5, v/v/w) as extraction solvent. This extraction procedure yielded plasma drug recoveries of greater than 60% and allowed UV detection at 220 nm without interference from endogenous components of plasma, urine or tissues. Linear standard curves up to 1.00 micrograms/ml and drug determination down to 0.01 microgram/ml were observed. This method has been successfully applied to the analysis of human plasma and urine samples and of encephales from tianeptine-dosed rats.     

Quantitative Oberflächenanalyse binärer Metallsysteme durch Ionen-Streuungs-Spektrometrie und Sekundärionen-Massenspektrometrie

For standard samples of some binary metal systems (Ag-Cu, Cu-Al, Cr-Ag) surface analyses are performed, by Ion-Scattering-Spectrometry (ISS) as well as by Secondary-Ion-Mass-Spectrometry (SIMS). The calibration curves are found to be bent. Linearization is achieved for ISS in a log-log plot by a special yet simple calibration method: the ‘method of binary ratios’ which renders possible a determination of concentrations c between 0,1 and 0,9 with a standard deviation of merely 0,05·(1?c). For SIMS linearization of the calibration curves could not be achieved. Instead a calibration function with only three parameters was deduced. It validity could be confirmed. The determination of concentrations by making use of this function yields a relative standard deviation of approximately 0,15 · (1?c). Both methods of surface analysis—ISS and SIMS—are therefore suitable to give results with high precision and accuracy-at least for binary metal systems.     

Characterization of high explosive particles using cluster secondary ion mass spectrometry

The use of secondary ion mass spectrometry (SIMS) for the detection and spatially resolved analysis of individual high explosive particles is described. A C(8) (-) carbon cluster primary ion beam was used in a commercial SIMS instrument to analyze samples of high explosives dispersed as particles on silicon substrates. In comparison with monatomic primary ion bombardment, the carbon cluster primary ion beam was found to greatly enhance characteristic secondary ion signals from the explosive compounds while causing minimal beam-induced degradation. The resistance of these compounds to degradation under ion bombardment allows explosive particles to be analyzed under high primary ion dose bombardment (dynamic SIMS) conditions, facilitating the rapid acquisition of spatially resolved molecular information. The use of cluster SIMS combined with computer control of the sample stage position allows for the automated identification and counting of explosive particle distributions on silicon surfaces. This will be useful for characterizing the efficiency of transfer of particulates in trace explosive detection portal collectors and/or swipes utilized for ion mobility spectrometry applications.     

Accurate analysis of trace earthy-musty odorants in water by headspace solid phase microextraction gas chromatography-mass spectrometry

A simple and sensitive method was developed for the simultaneous separation and determination of trace earthy-musty compounds including geosmin, 2-methylisoborneol, 2-isobutyl-3-methoxypyrazine, 2-isopropyl-3-methoxypyrazine, 2,3,4-trichloroanisole, 2,4,6-trichloroanisole, and 2,3,6-trichloroanisole in water samples. This method combined headspace solid-phase microextraction (HS-SPME) with gas chromatography-mass spectrometry and used naphthalene-d(8) as internal standard. A divinylbenzene/carboxen/polydimethylsiloxane fiber exposing at 90°C for 30 min provided effective sample enrichment in HS-SPME. These compounds were separated by a DB-1701MS capillary column and detected in selected ion monitoring mode within 12 min. The method showed a good linearity from 1 to 100 ng L(-1) and detection limits within (0.25-0.61 ng L(-1)) for all compounds. Using naphthalene-d(8) as the internal standard, the intra-day relative standard deviation (RSD) was within (2.6-3.4%), while the inter-day RSD was (3.5-4.9%). Good recoveries were obtained for tap water (80.5-90.6%), river water (81.5-92.4%), and lake water (83.5-95.2%) spiked at 10 ng L(-1). Compared with other methods using HS-SPME for determination of odor compounds in water samples, this present method had more analytes, better precision, and recovery. This method was successfully applied for analysis of earthy-musty odors in water samples from different sources.