Toxicological screening by liquid chromatography

Toxicological screening commonly requires analysis of biological specimens by a variety of colorimetric, immunological and chromatographic techniques. Gas chromatography and thin-layer chromatography are useful for simultaneous identification of multiple drug classes, but both methods require sample pretreatment. Recently, liquid chromatography has been applied to untreated urine and serum. A series of columns are utilized for sample purification, as well as efficient separation of drugs. Multiple wavelength UV detection provides sufficient data for qualitative identification of drugs and their metabolites.     

(Micellar) electrokinetic chromatography: an interesting solution for the liquid phase separation dilemma

High-performance liquid chromatography (HPLC) is a well-established method in modern analysis. The method is simple, very robust and is applicable to the majority of components to be analyzed in contrast to gas chromatography. Low efficiency and small peak capacity are sore points of HPLC when complex mixtures have to be separated. The reason for this dilemma is the small diffusion coefficient of the analytes in the liquid mobile phase compared to a gaseous phase. This review, complemented by exemplary calculated data and some latest results of our own research, illustrates the dilemma of liquid phase chromatography to achieve high efficiencies under reasonable conditions. It is shown that (micellar) electrokinetic chromatography, offering fast and efficient separations, is a very promising solution for this dilemma. Additional features of this method are possibilities of on-line analyte concentration, coupling to mass spectrometry and the easy change of selectivities by applying various separation additives. The pros and cons of electrokinetic chromatography are pointed out and some application examples are given.     

Simulated moving columns technique for chiral liquid chromatography

Enantioselectivity of chiral selectors is often relatively low in chiral HPLC. For difficult chiral separations, often only partial resolution is obtained rather quickly by column and mobile phase screening, and, by trial-and-error, additional method optimization is required to achieve complete resolution. This paper describes the development of a novel column-switching technique called "simulated moving columns" (SMC) to quickly achieve complete chiral resolution on columns with limited enantioselectivity. The simulated moving columns (SMC) technique uses two (2) or three (3) short chiral HPLC columns connected in series, and forces the unresolved enantiomers to recycle exclusively through the columns until sufficient resolution is attained. In effect, SMC helps to achieve chiral resolution by virtually multiplying the column length, thus enhancing separation efficiency and resolution, without increasing backpressure. Comparison of the standard non-SMC approach with SMC, and selected applications of chiral separations of pharmaceutical drug molecules are presented. Through measurement and calculation, evaluation of off-column band broadening resulting from a two-column SMC system is provided. The results clearly indicate that SMC eliminates the significant band broadening that is inevitable in the closed-loop recycling techniques currently used in preparative chromatography. Furthermore, SMC is not only useful to enhance resolution for analytical and preparative chiral separation, but also has great potential to enhance recovery and purity for difficult chiral preparative chromatography.     

Innovations in high-pressure liquid injection technique for gas chromatography: pressurized liquid injection system

In gas chromatography (GC), highly volatile liquefied hydrocarbons are commonly injected using devices such as high-pressure syringes, piston valves, liquid rotary sampling valves, or vaporizing regulators. Although these techniques are adequate in some cases, there are known deficiencies. A new generation of sampling valve has been recently innovated and commercialized. Some of the highlights of the pressurized liquid injection system (PLIS) include compact size, the capability to directly couple to an injection port without the need for preinjection vaporization and transfer lines, and sample sizes ranging from 0.2 to 2.0 micro L. Although the valve has a specification of helium leak-free rating of 82.7 bar (1200 psig), the valve passes a hydrostatic pressure test of up to 414 bar (6000 psig). In the unheated version of PLIS, vaporization of solutes occur mainly because of the sheering effect of carrier gas in combination with thermal energy drawn from an injection port or a heated adaptor. This was found to be adequate for solutes with high to medium volatility of up to nC14 hydrocarbon. A higher molecular weight range of up to nC44 hydrocarbon can be achieved with the implementation of a heated version of PLIS, in which the channel of the shaft can be resistively heated at a rate of up to 400 degrees C/s. With its first introduction in May 2002, PLIS has gained acceptance amongst practitioners in GC because it addresses a key unarticulated need in sample introduction/enrichment and by specifically targeting many deficiencies encountered in contemporary high-pressure injection devices. In this paper, the design and performance of the various valve systems of PLIS, as well as industrial chromatographic applications, is presented.     

Development of a broad toxicological screening technique for urine using ultra-performance liquid chromatography and time-of-flight mass spectrometry

Withdrawal of the support for the REMEDi HS drug profiling system has necessitated its replacement within our laboratories with an alternative broad toxicological screening technique. To this end, a novel method, based on ultra-performance liquid chromatography (UPLC) and time-of-flight (TOF) mass spectrometry, was developed for the routine analysis of urine samples. Identification was achieved by comparison of acquired data to libraries containing more than 300 common drugs and metabolites, and was based on a combination of retention time, exact mass and fragmentation patterns. Validation data for the method is presented and comprised an evaluation of the following parameters: precision; transferability of the methodology between the six collaborating laboratories; specificity; extraction recovery and stability of processed samples; matrix effects and sensitivity.This paper presents the benefits of supplementary fragmentation data with particular regard to increasing specificity and confidence of identification and its usefulness with overdosed samples. The utility of the method was assessed by the parallel analysis of 30 authentic urine samples using the REMEDi HS and UPLC-TOF. The latter provided enhanced detection, leading to the identification of twice as many drugs. Furthermore it did not miss any compounds that were identified by REMEDi HS. The UPLC-TOF findings were further verified by a combination of data from three other conventional screening techniques, i.e., GC-MS, HPLC-DAD and UPLC-MS/MS.     

Beta-induced fluorescence as a detection technique for liquid chromatography

The design of a beta-induced fluorescence detector based on a commercially available radioactive source is described. Results obtained with the detector attached to an hplc system operating in both normal and reverse phase modes are presented. The technique of quenched beta-induced fluorescence in normal phase chromatography is also described and some preliminary results presented.     

Micellar enzymology: methodology and technique

The methodological procedures employed both for serving investigations in the field of micellar enzymology and developed directly in micellar enzymology and being of general significance, such as an estimation of molecular masses and sizes of biocatalysts, titration of active sites of enzymes, chemical modification of proteins (enzymes), conjugation and nanogranulation, are reviewed. Potentialities of using and informativity of various techniques are analyzed.     

Micellar liquid chromatography retention model based on mass-action concept of micelle formation

Mass-action model of surfactant micelle formation has been used to develop a conceptual retention model in micellar liquid chromatography (MLC). The retention model bases on the consideration of the changes of the sorbate microenvironment at its transferring from the mobile phase (hybrid micellar eluent) to the stationary phase (a modified surface of alkyl-bounded sorbent). Principal retention equation contains the characteristics of hybrid micelles (critical micelle concentration, degree of counterion binding, partition coefficient of modifier between aqueous solution and micellar pseudo-phase) as well as three fitting parameters. The fitting parameters are an absolute term and coefficients that are equal to the number of molecules of surfactant and modifier, which are attached/detached by sorbate transferring from a hybrid micellar eluent to a modified surface of the stationary phase. On the MLC separation of five antibiotics of rubomicin derivatives and four esters of 4-hydroxybenzoic acid the model of the change of sorbate microenvironment has been tested. The adequateness of model to experimental data has been shown. A simple three-parameter function connecting log k with log cS and log cR that provides a high goodness-of-fit follows from principal retention equation (cS and cR are the molar concentrations of surfactant and organic modifier in the micellar eluent, respectively).     

Micellar liquid chromatography for lipophilicity determination of new biologically active 1,3‐purinodiones

A series of newly synthesized 1,3‐purinodiones with potential anticonvulsant activity, exhibiting affinity to adenosine A₁ and/or A_2A receptors, were subjected to micellar LC (MLC) with SDS as micelle‐forming agent and n‐propanol as organic modifier. Two C18 silica‐based columns were employed in MLC: a particle one and a monolithic. In parallel, those derivatives were also analyzed in RP‐LC on four silica‐based columns and on an immobilized artificial membrane column. The correlations between the relevant logarithms of the retention factors of analytes obtained in MLC, immobilized artificial membrane and RP‐LC systems on the one hand, and the calculated log P (clog P) and log D values (clog D) on the other, were examined. The level of the correlations of retention data from MLC and RP‐LC systems with clog P and clog D obtained is similar but it could be stressed that MLC allows increasing the speed of analysis and using only one mobile phase. Moreover, there is no need of applying an extrapolation procedure in lipophilicity determination. Therefore, the MLC systems, providing chromatographic data in a fast and efficient manner, were demonstrated as promising alternatives to the classical RP‐LC systems to estimate the lipophilicity of drugs and drug candidates.     

Micellar electrokinetic capillary chromatography for selected tropane alkaloid analysis in plant extract

Summary Micellar electrokinetic capillary chromatography was applied to the simultaneous analysis of six tropane alkaloids, including hyoscyamine and scopolamine. Successful results were obtained using a 30 mM boratephosphate buffer at basic pH (8.5) in the presence of 50 mM sodium dodecyl sulfate. The operating conditions, such as buffer concentration and pH, micelle concentration and organic modifier type and percentage were also discussed on the basis of the results given with a tropane alkaloid mixture. Addition of organic modifiers showed an improvement in separation efficiency and resolution. Moreover, hyoscyamine and littorine, two positional isomers, were only resolved by the addition of organic solvents such as methanol or acetonitrile. The optimized conditions were finally applied to the analysis of tropane alkaloids found in genetically transformed root cultures ofDatura candida x D. aurea. Dedicated to Professor Werner Haerdi on the occasion of his 70^th birthday.     

Determination of polynuclear aromatic hydrocarbons in environmental samples by Micellar liquid chromatography

A method for the separation of polycyclic aromatic hydrocarbons (PAHs) by high-performance liquid chromatography using a hybrid micellar mobile phase is described. The detection of PAHs was carried out using the fluorescence method with programmable excitation and emission wavelength. The method is applied to the analysis of several environmental samples (sea water, sediments, limpets, sea worms) and several of these compounds are quantitated at concentration below 70 ng L⁻¹(kg⁻¹) in the original samples.     

Micellar electrokinetic chromatography as a fast screening method for the determination of 1,4-dihydropyridine calcium antagonists

A micellar electrokinetic chromatographic method (MEKC) was optimized for the separation of six calcium antagonists. The effects of the buffer (concentration and pH), concentration of sodium dodecyl sulphate (SDS), the organic modifier, the injection time, and the voltage applied were studied. A final appropriate electrolyte of 50 mM borate buffer, pH 8.2, containing 20 mM SDS and 15% (v/v) acetonitrile was found to provide the optimum separation with respect to resolution and migration time. The samples were introduced hydrostatically for 4 s at 50 mbar injection pressure and the applied voltage was +25 kV. The screening of the six compounds was achieved in less than 15 min: nifedipine (migration time, tm = 6.9 min), nimodipine (tm = 10.1 min), felodipine (tm = 12.2 min), nicardipine hydrochloride (tm = 12.7 min), lacidipine (tm = 13.5 min) and amlodipine besylate (tm = 14.1 min, tm = 8 min). The method developed showed to be linear at least up to 70 micrograms/ml with a detection limit of about 5 micrograms/ml for each compound. The within-day and inter-day area reproducibility (R.S.D.) were, respectively, lower than 4.8 and 8.6% for six replicate samples.     

Micellar electrokinetic chromatography of ionic compounds: The analysis of hop bitter acids

Successful separation of the six major hop bitter compounds by micellar electrokinetic chromatography depends critically on precise pH control. Any factor which can affect the actual in-column pH must, therefore, also be carefully controlled: it has been found that temperature-induced changes of the buffer pH can cause loss of resolution and even reversals in elution order. The pH level at which the effects occur suggests that the pKa values of the analytes are modified by the micellar medium.     

Optimisation technique for stepwise gradient elution in reversed-phase liquid chromatography

An optimisation technique of reversed-phase liquid chromatographic separations based on gradient elution with a stepwise variation pattern of the volume fraction phi of the organic modifier in the water-organic mobile phase is presented. It uses a non-linear least-squares programme with a Monte-Carlo search for initial estimates in order to determine the best variation pattern that leads to the optimum separation of a mixture of solutes. The validity of the above methodology was tested by separating eight catechol-related solutes with mobile phases modified by methanol or acetonitrile and variation patterns of two, three or four steps in the psi values. It was found in all cases a very satisfactory accuracy of the predicted gradient elution times, which is of the same order with the accuracy of the retention times predicted under isocratic or linear gradient conditions. In addition, it was shown that the proposed optimisation technique is both effective and flexible but well-shaped chromatograms are obtained under electrochemical detection only if steps with increasing psi are used and the change in psi is programmed to occur at the intermediate of the predicted peaks.     

Comprehensive screening procedure for diuretics in urine by high-performance liquid chromatography

A rapid and reliable screening procedure using high-performance liquid chromatography for the detection of 23 diuretics (belonging to five different pharmacological groups) in urine has been developed. Two aliquots of 2-ml urine samples were extracted separately under acidic and basic conditions. The acidic and basic extracts were pooled, evaporated to dryness and reconstituted in methanol. The methanolic extract was injected onto a Hewlett-Packard Hypersil ODS C18 (5 microns) column (column I) and a Hewlett-Packard LiChrosorb RP-18 (5 microns) column (column II; an alternative column). The same gradient mobile phase was used for both columns. A diode array ultraviolet detector was set to monitor the signal to the integrator (Chem Station) at 230 and 275 nm. Recovery studies of the 23 diuretics were performed under acidic and basic conditions. The overall lower limits for detection on column I using both extraction procedures ranged from 0.5 to 1.5 micrograms/ml of urine (average 1.0 micrograms/ml). Amiloride, ethacrynic acid and probenecid could not be detected below 5 micrograms/ml of urine. No interference from the biological matrix was apparent. Amiloride could be detected in urine 4 h after oral administration of 15 mg of amiloride to a healthy volunteer, when the sample was extracted under alkaline conditions. The suitability of the screening method for the analysis of urine samples was tested by studying the variation with time of chlorthalidone, furosemide, probenecid, acetazolamide, quinethazone, spironolactone, bendroflumethiazide, bumetanide, triameterene and hydrochlorothiazide concentrations in the urine of normal human volunteers after minimum single or multiple (probenecid) doses.(ABSTRACT TRUNCATED AT 250 WORDS)