Luminarin 9: A new fluorescent reagent for the precolumn derivatizatioin of main volatile and non-volatile N-nitrosamines in RP-HPLC

Summary A sensitive precolumn fluorescence derivatization method for low level detection of the, volatile (N-nitrosodimethylamine and N-nitrosopyrrolidine) and non-volatile N-nitrosamines (N-nitrosoproline and N-nitrosodiethanolamine) an high-performance liquid chromatography was developed. This method is based on the denitrosation of the compounds of interest by a mixture of hydrobromic acid and acetic acid to produce the corresponding secondary amines. These are, then, able to react with, a quinolizinocoumarin derivative (luminarin 9^®) to form highly fluorescent labelled N-nitrosamine derivatives. The structural elucidation of the luminarin 9^® derivatives of N-nitrosoproline and N-nitrosodimethylamine by way of example, were established by liquid chromatography-mass spectrometry (LC-MS) and by direct chemical ionization-mass spectrometry (CI-MS). The separation, derivatization and detection conditioins were optimized for all the studied compounds. The detection limits (signal to noise ratio=3) were between 0.4 and 1.0 pmol injected depending on the compound. The calibration graphs were linear for derivatized amounts in the range of 0.5–40 nmol for N-nitrosodimethylamine and N-nitrosopyrrolidine, 0.4–2- nmol for N-nitrosoproline and 1.0–40 nmol for N-nitrosodiethanolamine. The repeatability (RSD less than 3.5%, n=6) and reproducibility (RSD less than 4.8%, n-6) were satisfactory.     

Postcolumn fluorescence as an alternative to evaporative light scattering detection for ceramide analysis with gradient elution in non-aqueous reversed-phase liquid chromatography

Ceramide analysis was developed with gradient elution in non-aqueous reversed-phase liquid chromatography with evaporative light scattering detection (ELSD) or postcolumn fluorescence detection. Fluorescence detection (excitation, 360 nm; emission, 425 nm) after postcolumn formation of mixed assemblies between eluted ceramides and 1,6-diphenyl-1,3,5-hexatriene was developed. In comparison with ELSD, fluorescence detection allows a better detection of the minor species ceramide from ceramide type III (commercial mixture of non-hydroxy fatty acid-sphingosine) and appears to be more sensitive for quantitation of ceramides at low concentrations. The fluorescence response is linear over a wide range of injected amount of ceramide III (expressed as stearoyl-phytosphingosine): 10 ng to 1000 ng. The response of ELSD is non linear but can be linearized in double logarithmic coordinates for calculations over a narrow range, e.g. between 10 to 350 ng ceramide III injected. The lower quantitation limits of these two detectors are similar: 5 ng ceramide III was injected.     

Application of the assay of aflatoxins by liquid chromatography with fluorescence detection in food analysis

HPLC using fluorescence detection has already become the most accepted method for the determination of aflatoxins due to its several advantages over other analytical methods. Both normal- and reversed-phase HPLC can be used. However the reversed-phase HPLC methods are more popular. Liquid chromatographic determination of aflatoxins using fluorescence detection and its application in food analysis is reviewed in this article.     

Direct determination of biacetyl in fats by sensitized room temperature phosphorescence

A direct method for the determination of biacetyl in butter and margarine by sensitized room temperature phosphorescence (SRTP) is described. After dissolution of the sample in hexane, biacetyl is isolated by distillation, and its native phosphorescence is sensitized by a non-polar linear furocoumarin, 4′5′-dihydro-3-carbethoxypsoralen. The limit of detection is 0.05 ng ml^?1 biacetyl, with a linear response from 1 × 10^?4 to 1 μg ml^?1 (r = 0.999). The RSD is 3.5% at 100 ng ml^?1.     

The benefit of the retrofitting of a conventional LC system to micro LC: a practical evaluation in the field of bioanalysis with fluorimetric detection

The interests in liquid micro-chromatography (higher column efficiencies, increase in sensitivity) are now well established. The enhancement of fluorimetric response induced by the reduction of the inner diameter of columns (4.6, 3.0, 1.0 and 0.3 mm respectively) coupled with adapted detection cells to control the loss of efficiency (8 micro L for the two first columns and 100 nL for the two smaller ones) has been studied in the bioanalytical field, using the plasma determination of native fluorescent antibacterial agents: fluoroquinolones. Ten-fold enhancement of the signal can easily be obtained when substituting a 0.3 mm i.d. column and 100 nL detection cell for a 4.6 mm i.d. column, and 8 micro L detection cell. In addition to inner diameter reduction, the detection cell geometry appears to be an essential parameter to obtain the best enhancement of the recorded signal. Hence, the enhancement of signal with micro-chromatography with fluorimetric detection appears to be a compromise between column inner diameter and flow cell volume reduction.     

Capillary electrophoresis for rapid identification of monoclonal antibodies for routine application in hospital

mAbs are widely used in cancer therapy. Their compounding, performed just before their administration to patients, is executed in a production unit of the hospital. Identification of these drugs, individually prepared in bags for infusion before patient administration, is of paramount importance to detect potential mistakes during compounding stage. A fast and reliable analytical method based on CZE combined to a cationic capillary coating (hexadimethrine bromide) was developed for identification of the most widely used compounded therapeutic for cancer therapy (bevacizumab, cetuximab, rituximab, and trastuzumab). Considering the high structural and physico‐chemical similarities of these mAbs, an extensive optimization of the BGE composition has been performed. The addition of perchlorate ions and polysorbate in the BGE greatly increased the resolution. To validate the method, an internal standard was used and the relative migration times (RTm) were estimated. Very satisfactory RSDs of the RTm for rituximab (0.76%), cetuximab (0.46%), bevacizumab (0.31%), and trastuzumab (0.60%) were obtained. The intraday and interday RSD of the method were less than 0.32 and 1.3%, respectively for RTm. Significant differences between theses RTms have been demonstrated allowing mAbs identification. Finally, accurate mAbs identification has been demonstrated by a blind test.     

Fluorescence and Mass Spectrometry Studies of Meta-tetra(hydroxyphenyl)chlorin Photoproducts

The meta-tetra(hydroxyphenyl)chlorin (m-THPC), a second-generation sensitizer used in photodynamic therapy (PDT), is currently under clinical trial. In vivo fluorometry provides direct evidence that photobleaching processes are induced at the tumor site during PDT. Photoproduct formation has thus to be taken into account to fully understand PDT treatment. A preliminary step is to determine the fluorescence characteristics of photoproducts formed in solution. Solutions of m-THPC irradiated at 514 nm have been separated by HPLC using absorption and fluorescence detection. Six main photoproducts have been isolated. According to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) results, five fluorescent photoproducts emitting at 652 nm have been attributed to three mono-, one di- and one tri-hydroxy derivatives (m/z 697, 713 and 729, respectively). Fluorescence characteristics of mono-hydroxy forms were found to be similar to those of m-THPC, whereas fluorescence yields in di- and tri-hydroxy derivatives were very low. Another product, corresponding to a MALDI-TOF MS main signal at m/z 542, showed an absorption spectrum maximum at 522 nm while a weak fluorescence was detected at 480 nm. The loss of the Soret band suggests that this photoproduct results from the opening of the reduced pyrrole ring. The part played by each of these products in the photobleaching phenomenon of m-THPC is discussed.     

Atmospheric pressure photoionization coupled to porous graphitic carbon liquid chromatography for the analysis of globotriaosylceramides. Application to Fabry disease

Globotriaosylceramides (Gb(3)) are biological compounds implicated in Fabry disease, a lysosomal storage disease due to the deficient activity of alpha-D-galactosidase A, which results in an accumulation of Gb(3) in many organs. The naturally occurring samples are composed of mixtures of several molecular species differing by the structure of the alkyl chains and the nature of the sphingoid base. Atmospheric pressure photoionization mass spectrometry (APPI-MS) proved to be an efficient method for the analysis of globotriaosylceramide molecular species, both in direct injection and by coupling with liquid chromatography (LC). In the positive ion mode, in-source fragmentations yield very precious information that can be used to determine the structure of the alkyl chains. In the negative ion mode, the chloroform solvent participates to the analyte ionization by forming an adduct with chloride ions generated in situ. Combination of LC on a Porous Graphitic Carbon stationary phase and APPI-MS allowed the detection of a great number of species from biological samples isolated from Fabry patients. This method could be an interesting analytical tool for the biochemical investigation of (sphingo) lipid metabolism.     

Investigation on non-optically bleachable components of ESR aluminium signal in quartz

Electron Spin Resonance (ESR) can be used as a method to estimate the age of sediment deposition using the paramagnetic centre related to aluminium impurities in quartz. This so-called Al-centre can be partially optically bleached and its signal intensity decreases in relation to time exposure to solar light, until it reaches a plateau value corresponding to a residual signal. This signal can be attributed to “Deep Aluminium Traps” (DAT) which cannot be reset by an exposure to sunlight. In this study, we have investigated the behaviour of the DAT signal in samples from different origins and ages. The intensity of the DAT signal has been isolated from the total aluminium signal by the exposure of different quartz samples to simulated solar light. We observed that the DAT intensities were sample dependant and therefore it should be determined for each sample. Moreover, DAT intensities of Pleistocene volcanic quartz increase with gamma laboratory irradiation, whereas DAT intensities of sedimentary quartz do not vary with added artificial doses. This suggests that DAT in quartz extracted from sediments must be inherited from the primary source of the quartz, and were saturated at the time of sedimentation. We thereby validate the ESR dating of quartz sediment protocol used so far.     

Simultaneous Quantification of Gentamicin and Colistin Sulfate in Pharmaceuticals using Ion-Pairing and Polarity Gradient Chromatography with Low-UV Detection

For the first time, a simple and rapid method for simultaneous determination of gentamicin sulfate and colistin sulfate in two pharmaceutical formulations for children and adults by ion-pairing reverse phase chromatography and low-UV detection at 215 nm has been developed. This simultaneous analysis is thus a challenge due to the multicomponent mixture of high polar, non volatile and non UV absorbing chromophores. Rapid separation required less than 5 min on a Waters X-Terra^® C18 MS column (50 mm × 4.6 mm i.d., 2.5 μm) with temperature maintained at 35 °C. A linear gradient from 15/85 to 40/60 acetonitrile/water (v/v) with constant hexafluorobutyric acid (HFBA) concentration of 0.05 % (v/v) was used as pairing reagent at 1.5 mL min⁻¹. In pharmaceutical analysis, the basic and polar compounds are separated by ion-pairing chromatography and the detection of analytes with weak chromophores requires working at low wavelengths. This application is an example of troubleshooting, i.e. baseline drift, due to gradient elution and absorbance of the ion-pairing agent. Baseline drift was minimized by optimizing the HFBA concentration gradient and its slope. Complete analytical validation was carried out according to the International Conference of Harmonization, and real samples were analyzed to demonstrate the applicability of the proposed method for routine use.