Profiling of impurities in illicit methamphetamine by high-performance liquid chromatography and capillary electrochromatography

High performance liquid chromatography (HPLC) with photodiode array (PDA) UV and fluorescence (FL) detection, and capillary electrochromatography (CEC) with laser-induced fluorescence (LIF) detection were investigated for the analysis of acidic extracts derived from illicit methamphetamine. These compounds include major impurities from the hydriodic acid/red phosphorous reduction method, i.e., 1,3-dimethyl-2-phenylnaphthalene and 1-benzyl-3-methylnaphthalene, and other trace-level, structurally related impurities. For certain of these solutes, HPLC with conventional FL detection gave at least a 60× increase in sensitivity over UV detection. In addition, other highly fluorescent impurities were detected in methamphetamine produced via four other synthetic routes. The use of a rapid scanning FL detector (with acquisition of “on the fly” excitation or emission) provided structural information and gave “optimum” excitation and emission detection wavelengths. CEC with LIF detection using UV laser excitation provided greatly improved chromatography over HPLC, with good detection limits in the low ng/ml range. Both methodologies provide good run-to-run repeatability, and have the capability to distinguish between samples.     

Profiling of impurities in heroin by capillary electrochromatography and laser-induced fluorescence detection

Capillary electrochromatography (CEC) with laser-induced fluorescence (LIF) detection was investigated for the analysis of acidic and neutral impurities in heroin. The phenanthrene-like heroin impurities exhibit high native fluorescence when excited with a doubled argon ion laser (operating at 257 nm). The limit of detection for acetylthebaol is 66 pg ml(-1). CEC-LIF analysis of heroin samples of different geographical origin gave distinguishable peak-enriched chromatograms. A sulfonic acid C12 polymer monolith column provided similar resolving power to a 1.5 mm non-porous ODS column for the isocratic analysis of a refined heroin sample. Analysis of a crude heroin sample via a multi-step gradient CEC resolved a significantly higher number of peaks than gradient high-performance liquid chromatography or micellar electrokinetic capillary chromatography.     

Determination of multiple drugs of abuse in human urine using capillary electrophoresis with fluorescence detection

Methods for separation and determination of multiple drugs of abuse in biological fluids using capillary electrophoresis (CE) with native fluorescence and laser-induced fluorescence (LIF) detection are described herein. Using native fluorescence, normorphine, morphine, 6-acetyl morphine (6-AM), and codeine were analyzed by CE without any derivatization procedure and detected at an excitation wavelength of 245 nm with a cut-off emission filter of 320 nm, providing a rapid and simple analysis. The detection limits were in the range of 200 ng/mL. For a highly sensitive analysis, LIF detection was also examined using a two-step precolumn derivatization procedure. In this case, drugs extracted from human urine were first subjected to an N-demethylation reaction involving the use of 1-chloroethyl chloroformate (ACE-Cl) and then derivatized using fluorescein isothiocyanate isomer I (FITC) and analyzed by CE coupled to a LIF detector. Variables affecting this derivatization: yield of demethylation reaction, FITC concentration, reaction time and temperature, were studied. The estimated instrumental detection limits of the FITC derivatives were in the range of 50-100 pg/mL, using LIF detection with excitation and emission wavelengths of 488 nm and 520 nm, respectively. The linearity, reproducibility and reliability of the methods were evaluated. In addition, a comparison of the characteristics for both native fluorescence and LIF detections was also discussed.     

Long-term effects of photodynamic therapy on fluorescence spectroscopy in the human esophagus.

Clinical interest in laser-induced fluorescence (LIF) spectroscopy and photodynamic therapy (PDT) is growing rapidly and may ultimately lead to close parallel use of these techniques. However, variations in LIF due to photosensitizer retention as well as tissue damage and healing processes may interfere with autofluorescence-based diagnostic methods. We have investigated the compatibility of these two techniques by quantifying PDT-induced changes in LIF in the human esophagus. Fluorescence spectra were collected endoscopically at excitation wavelengths (lambda ex) of 337, 400 and 410 nm in 32 patients. Measurements were performed immediately before and after PDT treatment with porfimer sodium and during follow-up procedures. In the months following PDT regions of reepithelialized squamous showed reduced autofluorescence in comparison with untreated squamous regions (P = 0.0007). Photosensitizer fluorescence was undetectable with lambda ex = 337 nm during follow-up procedures, whereas for lambda ex = 400 and 410 nm porfimer sodium fluorescence was noted for nearly a year after treatment. Therefore, residual photosensitizer fluorescence is likely to affect certain LIF-based diagnostic techniques during a period when patients are at high risk for tumor recurrence. Modification of LIF systems and/or the use of alternative photosensitizers may be required to optimize the detection of lesions in the post-PDT patient. Given the potential of LIF as a method for surveillance following cancer therapy, further investigation of the compatibility of specific LIF approaches with cancer pharmaceuticals may be warranted.     

Capillary electrophoresis-laser induced fluorescence-electrospray ionization-mass spectrometry: a case study

The simultaneous hyphenation of capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection and electrospray ionization-mass spectrometry (ESI-MS) as a novel combined detection system for CE is presented. beta-Carbolines were chosen as model analytes with a forensic background. Nonaqueous CE as well as conventional CE with an aqueous buffer system are compared concerning efficiency and obtainable detection limits. The distance between the optical detection window and the sprayer tip was minimized by placing the optical cell directly in front of the electrospray interface. Similar separation efficiencies for both detection modes could thus be obtained. No significant peak-broadening induced by the MS interface was observed. The high fluorescence quantum yield and the high proton affinity of the model analytes investigated resulted in limits of detection in the fg (nmol/L) range for both detection methods. The analysis of confiscated ayahuasca samples and ethanolic plant extracts revealed complementary selectivities for LIF and MS detection. Thus, it is possible to improve peak identification of the solutes investigated by the use of these two detection principles.     

Laser-Induced fluorescence detection in conventional-size liquid chromatography with a frequency-doubled argon-ion laser

Laser-induced fluorescence (LIF) detection in conventional-size column liquid chromatography is achieved at 257 nm with a frequency-doubled argon-ion laser. Short-wavelength excitation offers two important advantages: firstly, a wide variety of analytes can be excited, and secondly, the Raman scatter of the eluent does not interfere with the fluorescence of the analytes. A standard mixture of polynuclear aromatic hydrocarbons was studied, both with LIF detection and with a commercially available sensitive conventional fluorescence detector. The improvement in the detection limits ranges from about a factory of 4 to 30; the LIF detection limits are typically at the 50 ng l^?1 level, which corresponds to an injected amount of 0.5 pg.     

Some applications of near-ultraviolet laser-induced fluorescence detection in nanomolar- and subnanomolar-range high-performance liquid chromatography or micro-high-performance liquid chromatography

In this work we present some applications of near-UV laser-induced fluorescence (LIF) with micro-HPLC (microHPLC) and HPLC. To test the sensitivity of the detection, we used pyrene and aflatoxins, because both of these molecules exhibit native fluorescence. Then we studied catecholamines derivatized with 1,2-diphenylethylenediamine. The results show that we were able to reach better sensitivity levels than previously described in LIF studies. For catecholamines, a 50-fold increase in sensitivity compared to conventional fluorescence was obtained. These results indicate that LIF detection associated with HPLC or microHPLC can be used to detect very low concentrations of substances that can be excited in the near-UV range after labeling at nanomolar concentrations.     

Indirect laser-induced fluorescence detection of explosive compounds using capillary electrochromatography and micellar electrokinetic chromatography

Mixtures of nitroaromatic and nitramine explosive compounds and their degradation products were analyzed using electrokinetically driven separations with both indirect laser-induced fluorescence (IDLIF) and UV absorption detection. Complete separations of the 14-component mixture (EPA 8330) were achieved using both capillary electrochromatography (CEC) and micellar electrokinetic chromatography (MEKC). IDLIF detection was performed using an epifluorescence system with excitation provided by a 635 nm diode laser and micromolar concentrations of the dye Cy-5 as the visualizing agent. While the sensitivity of the two detection methods was similar for the nitroaromatic compounds, the nitramines could only be detected using UV absorption due to their low fluorescence quenching efficiency of Cy-5. The detection sensitivity using IDLIF was limited by low frequency oscillations in the fluorescence background. The oscillations increased with higher electric field strength and were attributed to thermal fluctuations caused by Joule heating. Due to the more conductive running buffer and higher separation currents used in MEKC, sensitive IDLIF detection could only be achieved using low (approximately 100 V/cm) field strengths, resulting in long analysis times. CEC separations, which are typically run with low conductivity mobile phases to avoid bubble formation, are less sensitive to this effect. In CEC separations with IDLIF detection a stable fluorescence background using Cy-5 could be established using only a nonporous stationary phase. In capillaries packed with porous silica particles, anomalous migration behavior was observed with charged dye molecules and a stable fluorescence background could not be established under electrokinetic flow. This is the first demonstration of IDLIF in packed channel CEC.     

Fluorescein-based pI markers for capillary isoelectric focusing with laser-induced fluorescence detection

We prepared a series of low-molecular-mass fluorescent ampholytes with narrow pI range. These fluorescein-based ampholytes are detection compatible with argon laser-induced fluorescence (LIF) detection. The selected properties, important for their routine use as fluorescent pI markers, were examined. The pI values of new fluorescein-based pI markers were determined by capillary isoelectric focusing (CIEF) using currently available low-molecular-mass pI markers for CIEF with photometric detection. The examples of CIEF with fluorometric detection of new compounds together with fluorescein isothiocyanate (FITC) derivatized proteins are presented.     

CEC and EKC of natural compounds

In this review, an overview of CEC and EKC methods with their developments are summarized for different natural compounds. It is divided into three main parts. The first part elaborates the separation of lipophilic compounds without any charged groups. The second part constitutes CEC and EKC of lipophilic compounds containing ionizable functional groups whereas the third part contains hydrophilic compounds. Packed, monolithic, coated, or raw fused-silica (FS) capillaries are among the choice for stationary phases. Applications of these phases on the above-mentioned three classes of compounds, coupled with different detection methods, e.g. MS or LIF, are explored and their advantages and disadvantages are discussed.     

Migration behavior and separation of active components in Glycyrrhiza uralensis Fisch and its commercial extract by micellar electrokinetic capillary chromatography

1-Anilinonaphthalene-8-sulfonic acid (1,8-ANS), 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) and 2-(p-toluidino)naphthalene-6-sulfonic acid (2,6-TNS) were evaluated as additives in different buffers for the detection of bovine whey proteins using laser-induced fluorescence (LIF) monitoring in capillary electrophoresis (CE). These N-arylaminonaphthalene sulfonates furnish a large fluorescence emission when associated to some proteins whereas their emission in aqueous buffers, such as those used in CE separations, is very small. To select the best detection conditions, the fluorescence of these probes was first compared using experiments carried out in a fluorescence spectrophotometer. Using bovine serum albumin (BSA) as a model protein, it was demonstrated that 2-(N-cyclohexylamino)ethanesulfonic acid (CHES) buffer (pH 8 and pH 10.2) and the fluorescent probe 2,6-TNS gave rise to the highest increase in fluorescence for BSA. When the composition of these separation buffers was optimized for the electrophoretic separations, CHES buffer, pH 10.2 was chosen as the most suitable buffer to detect bovine whey proteins. The limit of detection obtained for some whey proteins in CE separations was about 6.10(-8) M for BSA, 3.10(-7) M for beta-lactoglobulin A (beta-LGA), 3.10(-7) M for beta-lactoglobulin B (beta-LGB), and 3.10(-6) M for alpha-lactalbumin (alpha-LA). These detection limits were compared to those achieved using UV detection under the same separation conditions. The results showed that the detection limits of BSA, beta-LGA and beta-LGB were twice as good using LIF than with UV detection. However, the limit of detection for alpha-LA was better when UV was used. The applicability of LIF detection to CE separation of whey proteins in bovine milk samples was also demonstrated.     

Laser-Induced Fluorescence (LIF) Probe for <Emphasis Type="Italic">In-situ</Emphasis> Nitric Oxide Concentration Measurement in a Non-thermal Pulsed Corona Discharge Plasma Reactor

Laser-induced fluorescence (LIF) is an effective in-situ probe for NO concentrations below 300 ppm in a non-thermal plasma reactor. A new method has been developed to measure in-situ NO concentration in the reactor discharge region using a long-time—on the order of seconds—averaged fluorescence detection. This method, for quantifying NO concentration in a nonthermal plasma reactor, is simpler than a short-time—on the order of nanoseconds—fluorescence detection. For accurate measurement based on the new method, the LIF intensity must be close to the corona-induced fluorescence (CIF) intensity; the CIF intensity serves as a guide in selecting the LIF intensity. We find that a kinetic model proposed earlier works for two-tube reactors and represents the NO concentration in the middle of the reactor, which verifies the assumption of gas plug flow.     

Capillary gel electrophoresis of single-stranded DNA fragments with UV detection

Capillary gel electrophoresis has proven to be a powerful tool in biomedical research. We report our investigation of some of the critical parameters affecting separations of single-stranded DNA fragments as monitored by ultraviolet (UV) absorbance detection. Although not as sensitive as laser-induced fluorescence (LIF), UV absorbance detection allows one to calculate quite accurately, and inexpensively, the molarity of each separated DNA fragment and, moreover, the signal “fading” effect normally observed with LIF detection can be, in many cases, substituted for fluorescence to detect the many different single-stranded DNAs, as well as for detection of sequencing reactions.     

Laser-induced dispersed fluorescence detection of polycyclic aromatic compounds in soil extracts separated by capillary electrochromatography

Polycyclic aromatic hydrocarbons (PAHs) and nitrogen containing aromatic compounds (NCACs) are characterized in soil extracts and laboratory standards by capillary electrochromatography (CEC) with laser-induced dispersed fluorescence (LIDF) detection using a liquid-nitrogen cooled charge-coupled device detector. The LIDF detection technique provides information on compound identity and, when coupled with the high separation efficiencies of the CEC technique, proves useful in the analysis of complex mixtures. Differences in fluorescence spectra also provide a means of identifying co-eluting compounds by using deconvolution algorithms. Detection limits range from 0.5 to 96x10(-10) M for selected PAHs and 0.9-3.7x10(-10) M for selected NCACs. Soil extracts are also injected onto the CEC column to evaluate chromatographic method performance with respect to complex samples and the ability to withstand exposure to environmental samples.     

Choice of different dyes to label tyrosine and nitrotyrosine

In this work, we will present some attempts to analyze tyrosine and nitrotyrosine using capillary electrophoresis and either UV-Visible detection or laser-induced fluorescence (LIF) detection. An argon ion (488 nm) laser is used for fluorescein isothiocyanate (FITC) and 7-fluoro-4-nitro-2,1,3-benzoxadiazole (NBD-F). A near infrared (780 nm) laser is used for NIR 780 derivatives. The UV-Visible limit of detection is 2.5 microM whereas it is in the range of 30 nM for LIF detection.     

Laser-induced fluorescence detection at 266 nm in capillary electrophoresis. Polycyclic aromatic hydrocarbon metabolites in biota

The separation of five phenolic polycyclic aromatic hydrocarbon metabolites (hydroxy-PAHs) has been performed by cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) using a 30 mM borate buffer (pH 9.0) containing 60 mM sodium dodecyl sulfate and varying concentrations of gamma-cyclodextrin (gamma-CD). A concentration of 12.5 mM gamma-CD was found to provide a baseline separation of the five hydroxy-PAHs. We applied conventional fluorescence and laser-induced fluorescence (LIF) detection, using a new, small-size, quadrupled Nd-YAG laser emitting at 266 nm. The best limits of detection, in the low ng/ml range, were achieved using LIF detection. For all analytes, linearity was observed up to ca. 100 ng/ml. As an application, conjugated pyrene metabolites in hepatopancreas samples from the terrestrial isopods Oniscus asellus and Porcellio scaber were separated and detected. Finally, flatfish bile samples from individuals exposed to polluted sediment or crude oil, which were part of an interlaboratory study, were analyzed by CD-MEKC with conventional fluorescence and LIF detection to determine the 1-hydroxypyrene concentrations.     

Laser-induced fluorescence (LIF) recognition of the structural composition of porcine heart valves

Reconstruction and replacement of heart valves with grafts fro pig tissue is a common procedure. However, bioprosthetic valves wear out in a shorter time span than mechanical valves. Bioprosthetic valve structure may contribute to degenerative changes that lead to valve failure. There is, at present, no method to examine the structure of a tissue valve prior to implant. Laser-induced fluorescence (LIF) of natural fluorophores is an elegant method developed for the detection of tumors, dermal lesions and atherosclerosis. We have studied LIF as a potential diagnostic technique for analysis of valvular tissue. Using excimer laser excitation, we examined natural fluorescence recorded from porcine aortic, mitral and pulmonary valves. All three valve outflow surface tissue layers are less fluorescent at 390-450 nm than the inflow layers. Immunohistochemical analysis of collagen I and elastin content in inflow and outflow surface layers of all three valves correlated well with LIF intensities and dI/d lambda values at selected wavelengths. In conclusion, the differences observed in emitted LIF from valve surface layers are found to correlate well with diversity in the structural protein content. The LIF spectroscopic measurements may provide an appropriate tool for examination of tissue valve structure prior to use for implantation.     

Improving detection in capillary electrophoresis with laser induced fluorescence via a bubble cell capillary and laser power adjustment

Bubble cells have been frequently employed in capillary electrophoresis (CE) to increase the light path length with UV detection to provide an increase in the observed sensitivity of CE; however this approach has not been commonly used for laser-induced fluorescence detection (LIF) with CE. In this paper we study the influence of laser power on the sensitivity of detection in using conventional and enlarged fused silica capillaries for CE with LIF. When using the bubble cell capillary, the laser power must be decreased relative to use of the conventional capillary to reduce the effects of photodegradation of the species being illuminated by the laser. Even though the light intensity was decreased, an increase in sensitivity of detection was observed for most compounds when a bubble cell was used. This increase ranged from a factor of 8 for riboflavin (410 nm excitation) to 3.2 for most aromatic compounds (266 nm excitation), when using a 3x bubble cell compared with a conventional capillary. The bubble cell capillary was used for native detection of IgG by LIF at 266 nm. A limit of detection of 60 ng mL(-1) was obtained from a 20 pg injection, which was 40 times more sensitive than silver staining in conventional SDS/PAGE.     

Detection method for microchip separations

The features of analytical systems utilizing microfluidic devices, especially detection methods, are described. Electrochemical detection (EC), laser-induced fluorescence (LIF), mass spectrometry (MS), and chemical luminescence (CL) methods are covered. EC enables detection without labeling and has been used in recent years because of its low cost and sensitivity. LIF is the most generally used detection method in microchip separations. Use of LED as an excitation source for fluorescence measurement was also developed for the purpose of miniaturization of the entire system, including detection and separation. Although MS enables highly sensitive analysis, the interface between MS and micro channels is still under examination. This review with fifty-two references introduces interesting detection methods for microchip separations. Related separation methods using microfluidic devices are also discussed.     

Energy transfer in combustion diagnostics: experiment and modeling

Laser induced fluorescence (LIF) of OH (A 2 sigma +) is measured in several atmospheric-pressure flames using a short-pulse laser system (80 ps duration) in conjunction with an intensified streak camera. The two-dimensional signal-detection technique allows one to simultaneously monitor rotational and vibrational relaxation as well as electronic quenching. Rotationally-resolved LIF spectra affected by energy transfer are compared with the results of a rate-equation model and are found to be in reasonably good agreement. It is shown that a significant contribution of fluorescence detected by broad-band techniques is due to levels populated by vibrational energy transfer (VET). Implications for picosecond LIF techniques for the time-resolved, quench-free detection of OH are discussed. A detailed analysis is presented for fluorescence spectra originating from levels populated by VET after excitation of states in the OH (A 2 sigma +, v' = 2) level.