Rapid assay of cocaine, opiates and metabolites by gas chromatography—mass spectrometry

The simultaneous assay of cocaine, opiates and metabolites in small biological samples continues to be a difficult task. This report focuses upon tabulation of important techniques (extraction, derivatization, chromatographic conditions, detection mode, data acquisition) reported over the last decade that were used in the development of assays for these analytes. The most prevalent procedures for extraction of cocaine, opiates and metabolites were liquid—liquid and solid-phase extraction isolation methods. Following extraction analytes were derivatized and analyzed by gas chromatography—mass spectrometry. The technique most often used for chromatographic separation was fused-silica capillary column gas chromatography. Detection generally was performed by selected ion monitoring in the positive-ion electron-impact ionization mode, although full-scan acquisition and positive- and negative-ion chemical ionization methods have been used. It was apparent from the review that there is a continuing need for greater sensitivity and selectivity in the assay of highly potent opiates and for cocaine and metabolites.     

Determination of new 2,3-benzodiazepines in rat plasma using high-performance liquid chromatography with ultraviolet detection

A method for the analysis of [1-(4-aminophenyl)-3,5-dihydro-7,8-dimethoxy-4H-2,3-benzodiazepin-4-one] (CFM-2) and its analogues CFM-3, CFM-4 and CFM-5 in rat plasma was developed. The 2,3-benzodiazepines (2,3-BZs) were extracted by liquid–liquid extraction and analyzed using high-performance liquid chromatography (HPLC) with ultraviolet detection (UV) at 240 nm. The method exhibited a large linear range from 0.05 to 2 μg/ml with an intra-assay accuracy for all studied compounds ranging from 92 to 105.5%; whereas the intra-assay precision ranged from 0.59 to 8.16% in rat plasma. The inter-assay accuracy of CFM-2, CFM-4 and their 3-methyl derivatives, CFM-3 and CFM-5 ranged from 92.2 to 107% and the inter-assay precision ranged from 2.17 to 11.9% in rat plasma. The lower limit of detection was 5.5 ng/ml for CFM-2, 6.5 ng/ml for CFM-3, 7 ng/ml for CFM-4 and 8.5 ng/ml for CFM-5 in rat plasma. The pharmacokinetic study demonstrated that 2,3-BZs achieved a peak plasma concentration between 45 and 75 min after drug administration. Moreover, we observed that plasma chromatograms of rats treated with CFM-3, CFM-4 and CFM-5, respectively, showed a peak consistent with CFM-2. Our study suggests that CFM-4, CFM-5 and CFM-3 are prodrugs of CFM-2, in which they are biotransformed in vivo via different metabolic pathways. In particular, CFM-2 has been proven to possess anticonvulsant activity in various models of seizures, acting as α-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptor antagonist.     

High-performance liquid chromatographic determination of ambroxol in human plasma

Ambroxol has been determined in biological fluids using a rapid and sensitive high-performance liquid chromatographic method. The samples prepared from plasma by liquid—liquid extraction were analysed on reversed-phase silica gel by competing-ion chromatography with ultraviolet detection. The method was applied to the determination of ambroxol levels in twelve healthy volunteers after oral administration of 90 mg of ambroxol in tablets of Mucosolvan and Ambrosan.     

Advances in techniques for ecdysteroid analysis

Summary

Recent improvements in chromatographic methods for ecdysteroids are reviewed. Minor improvements in liquid chromatography have increased the separation and range of compounds which can be accomodated, some useful techniques have been introduced in thin layer chromatography, and with new radioactivity scanners and direct mass spectrometry from TLC, this technique retains an important place among the tools of the ecdysonist. Supercritical fluid chromatography has been suggested as a rapid and more sensitive alternative to liquid chromatography. In mass spectrometry, fast atom bombardment has proved most useful, though coupling HPLC through thermospray and ptasmaspray methods has been attempted, but as yet, these techniques lack sensitivity. New solid phase immunoassay methods using colorimetric rather than radioactivity measurements promise to give a better means of analysis of small amounts of ecdysteroids, capable of use in the presence of radiolabeled compounds. A solid phase separation of C-20, 22 diol ecdysteroids with immobilized phenylboronic acid has been reported.     

Separation methods for taurine analysis in biological samples

Taurine plays an important role in a variety of physiological functions, pharmacological actions and pathological conditions. Many methods for taurine analysis, therefore, have been reported to monitor its levels in biological samples. This review discusses the following techniques: sample preparation; separation and determination methods including high-performance liquid chromatography, gas chromatography, ion chromatography, capillary electrophoresis and hyphenation procedures. It covers articles published between 1990 and 2001.     

Sample preparation and high-resolution separation of mycotoxins possessing carboxyl groups

The chromatographic analysis of carboxyl-containing mycotoxins, such as fumonisin B₁, ochratoxin A, and citrinin, presents a continual challenge. Toxins must first be extracted from foods or tissues and then cleaned up before chromatographic separation and detection. Liquid–liquid extraction efficiencies for some carboxylic mycotoxins are marginal for spiked samples and uncertain for incurred residues. Immunoaffinity columns may be useful for concentrating mycotoxins from samples before chromatography. In almost every case, more than one analytical method must be used to confirm the identification of the mycotoxin. The fumonisins are especially troublesome to analyze because they are relatively insoluble in organic solvents, they are not separated easily by gas chromatography, and they do not respond to the usual absorbance or fluorescence detectors used in liquid chromatography. Fluorescence derivatization and electrospray liquid chromatography–mass spectrometry have now made it possible to detect trace levels of mycotoxins. The purity of mycotoxin standards for toxicological studies can be determined by liquid chromatography with either an evaporative light scattering detector or electrospray mass spectrometer. New developments in capillary electrophoresis, nonporous microsphere liquid chromatography, and detection methods for low-volatility compounds show promise for improving the analysis of mycotoxins in the future.     

Recent development of multi-dimensional chromatography strategies in proteome research

As a complementary approach to two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), multi-dimensional chromatography separation methods have been widely applied in all kinds of biological sample investigations. Multi-dimensional liquid chromatography (MDLC) coupled with bio-mass spectrometry (MS) is playing important roles in proteome research due to its high speed, high resolution and high sensitivity. Proteome analysis strategies mainly include bottom-up and top-down approaches which carry out biological sample separation based on peptide and protein levels, respectively. Electrophoretic methods combined with liquid chromatography like IEF-HPLC and HPLC-SDS-PAGE have been successful applied for protein separations. As for MDLC strategy, ion-exchange chromatography (IEX) together with reversed phase liquid chromatography (RPLC) is still a most widely used chromatography in proteome analysis, other chromatographic methods are also frequently used in protein pre-fractionations, while affinity chromatography is usually adopted for specific functional protein analysis. Recent MDLC technologies and applications to variety of proteome analysis have been achieved great development. A digest peptide-based approach as so-called "bottom-up" and intact protein-based approach "top-down" analysis of proteome samples were briefly reviewed in this paper. The diversity of combinations of different chromatography modes to set up MDLC systems was demonstrated and discussed. Novel developments of MDLC techniques such as high-abundance protein depletion and chromatography array were also included in this review.     

Quantification of endocannabinoids in rat biological samples by GC/MS: technical and theoretical considerations

In the last several years, interest has increased significantly about the endocannabinoids anandamide and 2-arachidonylglycerol, two lipid messengers that activate cannabinoid receptors. Quantification of these compounds in biological samples presents numerous technical challenges. Because of their low abundance, endocannabinoids are usually quantified by isotope dilution assays using mass spectrometry coupled to either gas chromatography or high-performance liquid chromatography. Although endocannabinoid levels in biological fluids, such as plasma and cerebrospinal fluid, can be directly determined by these techniques, the complex lipid profile of brain tissue samples mandates purification of lipid extracts before GC/MS analysis; this step is not necessary when using HPLC/MS. We have found that when silica gel chromatography is used for endocannabinoid purification, poor recovery and loss of deuterium from the internal standards lead to inaccurate estimation of endocannabinoid levels. By contrast, purification strategies using C(18) solid-phase extraction permits precise and reproducible GC/MS quantification of endocannabinoids in tissue samples.     

Flow‐based analysis using microfluidics–chemiluminescence systems

This review will discuss various approaches and techniques in which analysis using microfluidics–chemiluminescence systems (MF–CL) has been reported. A variety of applications is examined, including environmental, pharmaceutical, biological, food and herbal analysis. Reported uses of CL reagents, sample introduction techniques, sample pretreatment methods, CL signal enhancement and detection systems are discussed. A hydrodynamic pumping system is predominately used for these applications. However, several reports are available in which electro‐osmotic (EO) pumping has been implemented. Various sample pretreatment methods have been used, including liquid–liquid extraction, solid‐phase extraction and molecularly imprinted polymers. A wide range of innovative techniques has been reported for CL signal enhancement. Most of these techniques are based on enhancement of the mixing process in the microfluidics channels, which leads to enhancement of the CL signal. However, other techniques are also reported, such as mirror reaction, liquid core waveguide, on‐line pre‐derivatization and the use of an opaque white chip with a thin transparent seal. Photodetectors are the most commonly used detectors; however, other detection systems have also been used, including integrated electrochemiluminescence (ECL) and organic photodiodes (OPDs). Copyright © 2012 John Wiley & Sons, Ltd.     

Chromatographic methods for the determination of toxicants in faeces

Modern chromatographic techniques and their application in the determination of toxic compounds in faeces are reviewed. Faecal analysis may be of importance in toxicokinetic studies of xenobiotics in order to determine factors such as metabolism, body burden and major routes of elimination. Compounds of interest include various food constituents, drugs and occupational or environmental factors. Further, various mutagenic or carcinogenic compounds which are excreted by faeces have been indicated to represent risk factors for colorectal cancer. In this context, the chromatographic determination of the endogenously generated fecapentaenes and bile acids, both postulated etiological factors in colorectal carcinogenesis, is reviewed. For fecapentaene determination, several high-performance liquid chromatographic (HPLC) methods are available; however, the applicability of some of these methods is limited owing to insufficient separation of various isomeric forms or discrimination between fecapentaenes and their precursors. For the determination of bile acids in faeces, many chromatographic procedures have been reported, and the characteristics of the most relevant methods are compared and discussed. It is concluded that separation by gas chromatography (GC) in combination with mass spectrometry provides the highest selectivity and sensitivity. A relatively rapid alternative analysis for the determination of total and aqueous faecal bile acids is proposed. Further, methods for the determination of polycyclic aromatic hydrocarbons (PAHs) are reviewed. Although the use of radiolabelled PAHs in animal studies has many advantages, it cannot be applied for human biological monitoring and HPLC and GC provide sensitive alternatives. An HPLC method for the determination of non-metabolized PAHs in faeces is described.     

Solid-phase extraction or liquid chromatography coupled on-line with gas chromatography in the analysis of biological samples

This review provides an overview of the on-line coupling of solid-phase extraction or liquid chromatography with gas chromatography for the analysis of biological samples. Principles relevant to techniques are briefly presented and selected applications are described. Benefits of the coupled systems are discussed.     

Quantitation of vitamin B6 in biological samples by isotope dilution mass spectrometry

Methods have been developed for the simultaneous quantitative analysis of vitamin B6 forms in biological samples by isotope dilution mass spectrometry using deuterated forms of pyridoxine, pyridoxal, pyridoxamine, and pyridoxic acid. The biological fluid or tissue sample was homogenized and then treated with a cocktail containing appropriate amounts of each deuterated vitamer, as well as the deuterated, phosphorylated vitamer forms. The individual vitamers were isolated from the homogenate by a complex high-performance liquid chromatographic procedure that provided separate fractions for each of the six vitamers found in biological samples. Aldehydic B6 vitamers were reduced to the alcohol form prior to acetylation and analysis by gas chromatography/mass spectrometry (GC/MS). The three resulting vitamers were analyzed by electron ionization GC/MS using a silicone capillary column. The methods have been applied to analysis of vitamin B6 in liver, milk, urine, and feces at levels as low as 0.02 nmol/ml.     

A new on-line, in-tube pre-column derivatization technique for high performance liquid chromatographic determination of azithromycin in human serum

Pre-column derivatization methods for high performance liquid chromatographic assay of specific pharmaceutical agents using 9-fluorenylmethyl chloroformate (FMOC-Cl) have received special attention because highly fluorescent and stable adducts are provided by these methods. However, unlike the post-column on-line techniques, long derivatization time is needed and the reaction cannot be well controlled. A new, sensitive and fast pre-column on-line derivatization technique coupled with high-performance liquid chromatography using FMOC-Cl as labeling agent is described and validated for determination of azithromycin in human serum. After extraction of the drug from serum, the residue was reconstituted in mixture of acetonitrile-phosphate buffer (3:1, v/v; pH 8.5) and directly injected onto the chromatographic system. Continuous on-line derivatization and analysis of the compounds were successfully performed using in-tube elution of FMOC-Cl. The total time needed for derivatization and chromatographic analysis of the drug was 13 min. The assay was reliable and reproducible, with limit of quantification of 10 ng/ml. The described technique may offer significant advantages over existing off-line derivatization methods using FMOC-Cl.     

High-performance liquid chromatographic determination of biotin in biological materials after crown ether-catalyzed fluorescence derivatization with panacyl bromide.

This paper reports a high-performance liquid chromatographic (HPLC) technique to determine biotin in biological samples. Biotin and the internal standard dethiobiotin are converted into fluorescent derivatives by using panacyl bromide [p-(9-anthroyloxy)phenacyl bromide] as a fluorescence label. Biotin is extracted from biological tissue with trichloroacetic acid and the extract is purified by a combination of solid-phase extraction on C18 cartridges, ion-exchange chromatography on DOWEX formate resin, and thin-layer chromatography. The purified sample extract is derivatized in the presence of a crown ether. The resulting panacyl esters can be separated on reversed-phase as well as on normal-phase HPLC. Normal phase HPLC is preferable because it provides higher sensitivity and demands less sample pretreatment. Analysis of rat intestinal tissue revealed that only about 13% of the biotin is present in free form whereas 87% is bound in proteins from which it can be released by hydrolysis. Biotin values determined by this method are comparable to those obtained by other techniques.     

Survey of recent advances in analytical applications of immunoaffinity chromatography

Methods that use immunoaffinity chromatography (IAC) for sample preparation or detection are becoming increasingly popular as tools in the analysis of biological and nonbiological compounds. This paper presents an overview of immunoaffinity chromatography and examines some recent developments of this technique in analytical applications. The emphasis is placed on HPLC-based IAC methods or those that combine IAC with other instrumental techniques; however, novel approaches that employ low-performance IAC columns for chemical quantitation are also considered. Particular applications that are examined include (1) the use of IAC in the direct detection of analytes, (2) the extraction of samples by IAC prior to on- or off-line detection by other methods, (3) the use of IAC in chromatographic-based immunoassays, and (4) the development of postcolumn reactors based on IAC for the detection of analytes as they elute from other types of chromatographic columns. The advantages and limitations for each approach are considered. In addition, a summary is provided of reports in the literature that have used IAC for these various formats.     

Development of multidimensional liquid chromatography and application in proteomic analysis

As a complementary approach to 2D-PAGE, multidimensional liquid chromatography (MDLC) separation methods have been widely applied in all kinds of biological sample investigations. MDLC coupled with mass spectrometry is playing an important role in proteome research owing to its high speed, high resolution and high sensitivity. Among MDLC strategies, ion-exchange chromatography together with reversed-phase LC is still a most widely used chromatography in proteome analysis; other chromatographic methods are also frequently used in protein prefractionations. Recent MDLC technologies and applications to a variety of proteome analyses have achieved great development. The diversity of combinations of different chromatography modes to set up MDLC systems was demonstrated and discussed. Novel developments of MDLC techniques such as ultra-pressure system, array-based separation and monolithic material are also included in this article.     

A note on the losses of monosaccharides, amino sugars, and amino acids from extracts during concentration procedures

The reproducibility of recovery of components in organic extracts may be improved by the addition of a small quantity of glycerin prior to concentration techniques such as freeze-drying or rotary evaporation to dryness. Various biological and geological samples have been analyzed for three different groups of natural hydrophilic organics using modern liquid chromatographic systems in order to examine this effect in detail.     

Development of multidimensional liquid chromatography and application in proteomic analysis

As a complementary approach to 2D-PAGE, multidimensional liquid chromatography (MDLC) separation methods have been widely applied in all kinds of biological sample investigations. MDLC coupled with mass spectrometry is playing an important role in proteome research owing to its high speed, high resolution and high sensitivity. Among MDLC strategies, ion-exchange chromatography together with reversed-phase LC is still a most widely used chromatography in proteome analysis; other chromatographic methods are also frequently used in protein prefractionations. Recent MDLC technologies and applications to a variety of proteome analyses have achieved great development. The diversity of combinations of different chromatography modes to set up MDLC systems was demonstrated and discussed. Novel developments of MDLC techniques such as ultra-pressure system, array-based separation and monolithic material are also included in this article.     

Analytical methods for the quantitative determination of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors in biological samples

Published analytical methods for the quantitative determinations of presently available five 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors ("statins"), lovastatin, simvastatin, pravastatin, fluvastatin and atorvastatin, are reviewed for therapeutic drug monitoring purpose in patients. Almost all assay reviewed are based on high-performance liquid chromatography or gas chromatography. Some purification steps (liquid-liquid extraction, solid-phase extraction, etc.) have been used before they are submitted to separation by chromatographic procedures and they are detected by various detection methods like UV, fluorescence and mass spectrometry. This review shows that most method may be used quantitative determination of statins in plasma and they are suitable for therapeutic drug monitoring purpose of these drugs.     

Specific and sensitive quantitation of 2,2′-dichlorodiethyl sulphide (sulphur mustard) in water, plasma and blood: application to toxicokinetic study in the rat after intravenous intoxication

A sensitive and specific capillary gas chromatographic method has been developed to measure trace amounts of 2,2′-dichlorodiethyl sulphide (sulphur mustard) in environmental or biological samples. Sulphur mustard was isolated from water or plasma by a solid-phase extraction procedure and from blood by liquid—liquid extraction. The accuracy and precision of the methods were demonstrated using replicate analyses of spiked water, plasma or blood: within-run and between-run variabilities were less than 20%. These analytical methods were used to evaluate the rate of sulphur mustard degradation in water or plasma. Good linear calibration curves, with a detection limit of 45 ng/ml, were obtained for quantitation and determination of sulphur mustard in blood following its intravenous administration to rats. Initial toxicokinetic data were obtained.