HPLC of 9-fluorenylmethylchloroformate-polyamine derivatives with fluorescence detection

Summary There are a number of reagents available for fluorescent labelling of primary amines. These include dansyl chloride, o-phthalaldehyde, fluorescamine, and a new reagent, 9-fluorenylmethylchloroformate (FMOC), reported recently. This paper describes a reversed-phase HPLC procedure for the separation and fluorescence detection of polyamines following pre-column derivatization with FMOC. The polyamines studied by this method include putrescine, cadaverine, spermidine, and spermine. Experiments were carried out to determine maximum fluorescence excitation and emission wavelengths, optimum reaction pH, linear ranges, and minimum detection limits for each of the polyamines. The HPLC method includes a gradient program which provides complete separation from serum hydrolysate components and specificity for the four polyamines with detection limits ranging from 2 to 9 pg. This procedure was applied to hydrolyzed serum samples.     

Spectrofluorimetric determination of moxifloxacin in tablets, human urine and serum

A spectrofluorimetric method to determine the antibiotic moxifloxacin is proposed and was applied to pharmaceuticals, human urine and serum. The fluorimetric method allows the determination of 30-300 ng mL-1 moxifloxacin in aqueous solution containing phosphoric acid-phosphate buffer (pH 8.3) with lambda exc = 287 nm and lambda em = 465 nm. Detection and quantification limits were 10 and 30 ng mL-1, respectively, with a relative standard deviation (n = 10) of 2%. This method was applied to the determination of moxifloxacin in three Spanish commercial pharmaceutical formulations. Another variant of the method in micellar medium allows the direct measurement of moxifloxacin in human serum and urine by standard additions. The enhanced fluorescence of moxifloxacin in 8 mM sodium dodecyl sulfate (SDS) solution at pH 4.0 (acetic acid-acetate buffer) for lambda exc = 294 nm and lambda em = 503 nm shows the same linear range as the aqueous method with a 25% lower slope (with detection and quantification limits of 15 and 60 ng mL-1, respectively, and a relative standard deviation of 1.3%), but permits the background fluorescence for urine and serum blanks to be minimized. Hence, sufficient sensitivity is reached to determine therapeutic concentrations of the drug in urine (average recovery 102 +/- 2%) and serum (average recovery 105 +/- 2%) samples.     

Capillary zone electrophoresis applied to the determination of the angiotensin-converting enzyme inhibitor cilazapril and its active metabolite in pharmaceuticals and urine

A capillary zone electrophoresis method has been developed for the quantitation of antihypertensive drug cilazapril and its active metabolite cilazaprilat in pharmaceuticals and urine. The separation of the compounds was performed in a fused-silica capillary filled with the running electrolyte, which consisted of a 60 mM borate buffer solution at pH 9.5. Under the optimized experimental conditions, the separation took less than 5 min. The analysis of urine samples required a previous solid-phase extraction step using C8 cartridges. The method was successfully applied to the determination of the drug and its metabolite in urine samples obtained from three hypertensive patients (detection limits of 115 ng ml(-1) for cilazaprilat and 125 ng ml(-1) for cilazapril) and to pharmaceutical dosage forms. The method was validated in terms of reproducibility, linearity and accuracy.     

Sensitive determination of methylenedioxylated amphetamines by liquid chromatography

Different strategies for the liquid chromatographic determination of methylenedioxylated amphetamines were evaluated: separation and detection of underivatized analytes by (i) UV or (ii) fluorescence, (iii) derivatization with 3,5-dinitrobenzoyl chloride followed by separation and UV detection of the derivatives formed and (iv) derivatization with 9-fluorenylmethyl chloroformate (FMOC) and subsequent separation and fluorimetric detection of the derivatives. The compounds tested were 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyethylamphetamine (MDE). On the basis of these studies, a new procedure for the chromatographic determination of MDA, MDMA and MDE is proposed, based on derivatization with FMOC. The described procedure allows the quantification of the tested compounds with adequate linearity, reproducibility and accuracy in the concentration interval 0.5-20.0 micrograms mL-1. The limits of detection were 0.01 microgram mL-1 for MDA and 0.025 microgram mL-1 for MDMA and MDE. The utility of the described assay was tested by determining methylenedioxylated amphetamines in plasma and urine.     

Analysis of lamotrigine and its metabolites in human plasma and urine by micellar electrokinetic capillary chromatography

A reliable micellar electrokinetic capillary chromatographic method was developed and validated for the determination of lamotrigine and its metabolites in human plasma and urine. The variation of different parameters, such as pH of the background electrolyte (BGE) and Sodium dodecyl sulfate (SDS) concentration, were evaluated in order to find optimal conditions. Best separation of the analytes was achieved using a BGE composed of 10 mM borate and 50 mM SDS, pH 9.5; melatonin was selected as the internal standard. Isolation of lamotrigine and its metabolites from plasma and urine was accomplished with an original solid-phase extraction procedure using hydrophilic-lypophilic balance cartridges. Good absolute recovery data and satisfactory precision values were obtained. The calibration plots for lamotrigine and its metabolites were linear over the 1-20 microg/mL concentration range. Sensitivity was satisfactory; the limits of detection and quantitation of lamotrigine were 500 ng/mL and 1 microg/mL, respectively. The application of the method to real plasma samples from epileptic patients under therapy with lamotrigine gave good results in terms of accuracy and selectivity, and in agreement with those obtained with an high-performance liquid chromatography (HPLC) method.     

Solid-phase extraction and determination of dansyl derivatives of unconjugated and acetylated polyamines by reversed-phase liquid chromatography: improved separation systems for polyamines in cerebrospinal fluid, urine and tissue

A sensitive and simple liquid chromatographic assay with fluorometric detection for unconjugated and acetylated polyamines in biological fluids is described. After precolumn derivatization with dansyl chloride, unconjugated polyamines and acetylated polyamines were extracted by elution from a Bond-Elut C18 column and then separated on a reversed-phase column with gradient elution. The complete analysis of unconjugated putrescine, spermidine, and spermine in either hydrolyzed urine, cerebrospinal fluid or tissue could be accomplished within 20-26 min, while the simultaneous analysis of unconjugated polyamines and monoacetylpolyamines could be completed within 40 min. Unhydrolyzed urine and cerebrospinal fluid required a Bond-Elut cation-exchange clean-up before dansylation. Standard curves for the assay were linear up to 20 nmol/ml, and the within-day and day-to-day coefficients of variation were between 1.1 and 4.6% and between 1.6 and 11.8%, respectively. Results obtained with the method were compared with results obtained with a well established modified amino acid analyzer method for urine, tissue and cerebrospinal fluid samples. The correlation coefficients between these two methods were in the range 0.933-0.996. Detection limits between 50 and 150 fmol were achieved for unconjugated and acetylated polyamines. Of more than twenty drugs and amines tested for possible interference with the assay, only normetanephrine was found to have the same retention time as the internal standard 1,6-diaminohexane.     

Determination of type A trichothecenes by high-performance liquid chromatography with coumarin-3-carbonyl chloride derivatisation and fluorescence detection

A method for the analysis of type A trichothecenes T-2 toxin, HT-2 toxin, neosolaniol and diacetoxyscirpenol by high-performance liquid chromatography with fluorescence detection using coumarin-3-carbonyl chloride has been developed. Different parameters concerning the analytical procedure such as stability of both the reagent and derivatised analytes, time and temperature of the derivatisation reaction, were studied and optimised. Three different clean-up procedures (solid-phase extraction with silica gel or C-18 cartridges, and liquid–liquid partition between toluene and dihydrogen phosphate buffer) were tested in order to remove the excess reagent peaks. The last procedure gave the best results when the buffer pH was 3–5.5, and is therefore recommended. Separations were performed on a stainless steel LiChrospher 100 C-18 reversed-phase column with pre-column of the same phase. The mobile phase was acetonitrile/water (65:35, v/v) containing 0.75% acetic acid at a flow-rate of 1.0 ml/min. The proposed method provides good separation between the four trichothecenes and good reproducibility (RSD of calibration standards <5%). The limits of detection of the studied trichothecenes at a signal-to-noise ratio of 3:1, with an injection volume of 20 μl were 10 ng/g sample for T-2 toxin and about 15 ng/g sample for the remaining mycotoxins. The calibration curve was linear between 10 and 2000 ng for the four trichothecenes assayed. The method was applied to the analysis of these mycotoxins in fungal cultures (corn and rice) of Fusarium sporotrichioides, and is also perfectly suitable for the quantification of type A trichothecenes in contaminated cereals.     

Sensitive determination of aliphatic amines in water by high-performance liquid chromatography with chemiluminescence detection

A sensitive method has been developed for liquid chromatographic determination of short aliphatic amines in water samples. Analytes are preconcentrated and dansylated on solid sorbents (C18 solid-phase extraction cartridges). The dansyl derivatives are chromatographed and post-column mixed with peroxyoxalate (TCPO) and H2O2 in order to perform chemiluminescence detection. Optimal results have been obtained using a sample volume of 5 ml. The method has been applied to the quantification or screening of several aliphatic amines: methylamine, ethylamine, butylamine, diethylamine, pentylamine and hexylamine. The screening procedure has been developed including also polyamines (putrescine, cadaverine, spermidine and spermine). The results obtained by using chemiluminescence (CL) detection have been compared with other detection systems (fluorescence and UV). The sensitivity can increase from 3 to 75 times respect UV detection and from 2 to 10 times respect fluorescence detection depending on the amine. The detection limits achieved were between 0.15 and 0.9 microg/l.     

Combination of hollow fiber-based liquid-phase microextraction with sweeping techniques in micellar electrokinetic chromatography for the determination of Strychnos alkaloids in human urine

A new method for the enrichment of Strychnos alkaloids in biological samples via liquid-phase microextraction (LPME) based on porous polypropylene hollow fibers in combination with on-line sweeping in micellar electrokinetic chromatography was developed. The calibration curve was linear over the range of 20-200 ng mL-1 for both strychnine and brucine in human urine sample. The detection limits (S/N=3:1) for strychnine and brucine were 1 ng mL-1 and 2 ng mL-1, respectively. The LPME-sweeping method has been successfully applied to the analysis of strychnine and brucine in real urine samples.     

Determination of the angiotensin-converting enzyme inhibitor quinapril and its metabolite quinaprilat in pharmaceuticals and urine by capillary zone electrophoresis and solid-phase extraction

Quinapril is an antihypertensive drug commonly used in the treatment of hypertension and congestive heart failure. In this work, a capillary zone electrophoresis system is optimized for the analysis of quinapril and its active metabolite quinaprilat in urine, as well as for the determination of the drug and its combination with hydrochlorothiazide in pharmaceuticals. The separation takes place in a fused-silica capillary. The running electrolyte consists of a 60 mM borate buffer solution, pH 9.5. The analysis of urine samples requires a previous extraction step using C8 solid-phase cartridges. Under the optimum experimental conditions, the separation of the two analytes and the internal standard takes less than 5 min. The detection limits obtained (75 and 95 ng/mL for quinapril and quinaprilat, respectively) allow the application of the electrophoretic method to the determination of the drug and its metabolite in urine samples obtained from four patients treated with quinapril.     

Solid-phase extraction with liquid chromatography and ultraviolet detection for the assay of antidepressant drugs in human plasma

A solid-phase extraction (SPE) method for sample clean-up followed by a reversed-phase high-performance liquid chromatography (HPLC) procedure for the assay of five antidepressant drugs (trazodone, doxepin, desipramine, maprotiline and imipramine) is reported. The drugs were recovered from plasma buffered at a suitable pH using C18 Bond-Elut cartridges and mixtures of methanol-aqueous buffer as washing and elution solvents. The recoveries of the drugs using other sorbent materials (C8, C2, cyclohexyl, cyanopropyl and phenyl Bond Elut and copolymer HLB waters cartridges) were also examined. The selectivity of SPE was examined by using spiked plasma samples and the CH cartridge gave rise to the cleanest extracts. Cyclohexyl cartridges were conditioned successively with 2 ml of methanol and 1 ml of acetic acid-sodium acetate buffer (0.1 M, pH 4.0). Plasma sample was buffered at pH 4.0 and then applied to the sorbent. The washing step was performed subsequently with 1.5 ml of acetate buffer (0.1 M, pH 4.0), 100 microl of acetonitrile and 1 ml of methanol-acetate buffer (30:70, v/v). Finally, the analytes were eluted with 0.5 ml of methanol-acetate buffer (70:30, v/v). The extract was evaporated to dryness, reconstituted in mobile phase, and chromatographed on a reversed-phase C18 column with ultraviolet detection at 215 nm. The recoveries of trazodone, doxepin, desipramine, maprotiline and imipramine from spiked plasma samples using the CH cartridge were 58 2, 84 3, 83 3, 83 3 and 82 2%, respectively. The within-day and between-day repeatabilities were lower than 6% and 9%, respectively. The linearity of calibrations for the five antidepressants was between 0.005 and 2 microg/ml. The limits of detection were 1 ng/ml for trazodone, doxepin and desipramine and 2 ng/ml for maprotiline and imipramine.     

Determination of sulphonate dyes in water by ion-interaction high-performance liquid chromatography

An ion-interaction high-performance liquid chromatography method for quick separation and determination of the sulphonated dyeAcid Yellow 1, and the sulphonated azo dyes Acid Orange 7, Acid Orange 12, Acid Orange 52, Acid Red 2, Acid Red 26, Acid Red 27 and Acid Red 88 has been developed. An RP-ODS stationary phase is used, and the mobile phase contains an acetonitrile-phosphate buffer (27:73, v/v) mixture at pH 6.7, containing 2.4 mM butylamine as ion-interaction reagent. Good separations were obtained using isocratic elution and spectrophotometric detection at 460 nm. The detection limits for the eight dyes ranged from 7 to 28 microg/l for an injection volume of 100 microl. Spiked tap water samples (100 ml), containing different concentration levels (0.3-1.2 microg/l) of the dyes were analyzed after acidification (pH 3) and preconcentration in disposable solid-phase extraction C18 cartridges.     

Determination of quinolones in chicken tissues by liquid chromatography with ultraviolet absorbance detection

This paper presents an analytical method for the determination of quinolones in chicken tissues. The procedure involves pre-treatment by solid-phase extraction (SPE) and subsequent liquid chromatography (LC) with UV absorbance detection. Different SPE disposable cartridges and extractants of the tissue samples were tested, and various columns were systematically tested. The mobile phase was composed of acetonitrile and citric buffer at pH 4.5, with an initial composition of acetonitrile-water (12:88, v/v) and using linear gradient elution. Recoveries were 66-91% in the concentration range 30-300 microg kg(-1). The detector response was linear in this range. The limits of detection were 16-30 microg kg(-1). These values were lower than the maximum residue limits established by the European Union.     

Simultaneous analysis of classical neuroleptics, atypical antipsychotics and their metabolites in human plasma

A high-performance liquid chromatographic method has been developed for the simultaneous determination of classical neuroleptics (chlorpromazine, haloperidol, loxapine and clotiapine), atypical antipsychotics (clozapine, quetiapine and risperidone) and their active metabolites (N-desmethylclozapine, clozapine N-oxide and 9-hydroxyrisperidone) in human plasma. Separation was obtained by using a C8 reversed-phase column and a mobile phase composed of 70% aqueous phosphate buffer containing triethylamine at pH 3.0 and 30% acetonitrile. The UV detector was set at 238 nm and amitriptyline was used as the internal standard. A careful pre-treatment procedure of plasma samples was developed, using solid-phase extraction with cyanopropyl cartridges, which gives high extraction yields (>or=93%). The limits of quantitation (LOQ) were always lower than 2.6 ng mL-1 and the limits of detection (LOD) were always lower than 0.9 ng mL-1 for all analytes. The method was applied with success to plasma samples from schizophrenic patients undergoing polypharmacy with two or more different antipsychotics. Precision data and accuracy results were satisfactory and no interference from other central nervous system (CNS) drugs was found. Hence the method is suitable for the therapeutic drug monitoring (TDM) of the analytes in psychotic patients' plasma.     

Simultaneous determination of doxorubicin, daunorubicin, and idarubicin by capillary electrophoresis with laser-induced fluorescence detection

The separation and simultaneous determination of doxorubicin, daunorubicin and idarubicin was investigated using capillary electrophoresis with laser-induced fluorescence detection. Because the three anthracycline antibiotics were similar in structure and mass, careful manipulation of the electroosmotic flow and electrophoretic mobilities was required. A buffer consisting of 100 mM borate, adjusted to pH 9.5, containing 30% acetonitrile was found to provide a very efficient and stable electrophoretic system for the analysis of the three anthracyclines. The method was applied to the determination of three anthracyclines in serum samples. Responses were linear in the range of 10-500 ng.mL-1 and the detection limits were lower than 0.9 ng.mL-1.     

On-line coupling of solid-phase extraction with mass spectrometry for the analysis of biological samples. II. Determination of clenbuterol in urine using multiple-stage mass spectrometry in an ion-trap mass spectrometer

Solid-phase extraction (SPE) was coupled to ion-trap mass spectrometry to determine clenbuterol in urine. For SPE a cartridge exchanger was used and, after extraction, the eluate was directly introduced into the mass spectrometer. For two types of cartridges, i.e. C18 and polydivinylbenzene (PDVB), the total SPE procedure (including injection of 1 mL urine, washing, and desorption) has been optimised. The total analysis, including SPE, elution, and detection, took 8.5 min with PDVB cartridges, while an analysis time of 11.5 min was obtained with C18 cartridges. A considerable amount of matrix was present after extraction of urine over C18 cartridges, resulting in significant ion suppression. With PDVB cartridges, the matrix was less prominent, and less ion suppression was observed. For single MS, a detection limit (LOD) of about 25 ng/mL was found with PDVB cartridges. With C18 cartridges an LOD of only about 50 ng/mL could be obtained. Applying tandem mass spectrometry (MS/MS) did not lead to an improved LOD due to an interfering compound. However, a considerable improvement in the LOD was obtained with MS3. The selectivity and sensitivity were increased by the combination of efficient fragmentation of clenbuterol and reduction of the noise. Detection limits of 2 and 0.5 ng/mL were obtained with C18 and PDVB cartridges, respectively. The ion suppression was 4 to 45% (concentration range: 250 to 1.0 ng/mL) after extraction of urine using PDVB cartridges, and up to 70% ion suppression was observed using C18 cartridges. With MS4, no further improvement in selectivity and sensitivity was achieved, due to inefficient fragmentation of clenbuterol and no further reduction of noise.     

Thousand fold concentration of an alkaloid in capillary zone electrophoresis by micelle to solvent stacking

In this paper, the co-solvent of methanol-water was used to facilitate the sodium dodecyl sulfate (SDS) micelles collapse, thereby inducing the on-line sample focusing technique of micelle to solvent stacking (MSS). To demonstrate this stacking method, the mechanism of micelles collapse in co-solvent was discussed. The details of the required conditions were investigated and the optimized conditions were: running buffer, 20mM H(3)BO(3) and 20mM NaH(2)PO(4) solution (pH 4.0); micellar sample matrix, 20mM SDS, 20mM H(3)BO(3) and 20mM NaH(2)PO(4) solution (pH 4.0); co-solvent buffer, 20mM H(3)BO(3) and 20mM NaH(2)PO(4) in methanol/water (90:10, v/v). The validity of the developed method was tested using cationic alkaloid compounds (ephedrine and berberine) as model analytes. Under the optimized conditions, this proposed method afforded limits of detection (LODs) of 0.5 and 1.1ng/mL with 300 and 1036-fold improvements in sensitivity for ephedrine and berberine, respectively, within 15min.     

Determination of oxytetracycline and some impurities in plasma by non-aqueous capillary electrophoresis using solid-phase extraction

Summary The potential of a non-aqueous, capillary electrophoresis (NACE) system for separating oxytetracycline from three of its impurities—tetracycline, 4-epioxytetracycline and 4-epitetracycline—using UV detection has been studied. The running buffer was: 25 mM sodium acetate, 1 mM EDTA, methanesulfonic acid, pH 4, dissolved in MeOH-ACN (50∶50,v/v). The method was also used to determine these compounds in pig plasma. A solid-phase extraction (SPE) procedure as a clean-up step has also developed. For this we tested Sep Pak C₁₈, LiChrolut EN and OASIS cartridges. OASIS cartridges were best. Recoveries were 90–100% for all compounds except EOTC which had a recovery of 74%.     

Migration behaviour and separation of tramadol metabolites and diastereomeric separation of tramadol glucuronides by capillary electrophoresis

Capillary electrophoresis with UV detection was used to separate tramadol (TR), a centrally acting analgesic, and its five phase I (M1, M2, M3, M4, M5) and three phase II metabolites (glucuronides of M1, M4 and M5). Several factors were evaluated in optimisation of the separation: pH and composition of the background electrolyte and the influence of a micellar modifier, sodium dodecyl sulfate. Baseline separation of TR and all the analytes was obtained with use of 65 mM tetraborate electrolyte solution at pH 10.65. The lowest concentrations of the analytes that could be detected were below 1 microM for the O-methylated, below 2 microM for the phenolic and ca. 7 microM for the glucuronide metabolites. The suitability of the method for screening of real samples was tested with an authentic urine sample collected after a single oral dose (50 mg) of TR. After purification and five-fold concentration of the sample (solid-phase extraction with Oasis MCX cartridges), the parent drug TR and its metabolites M1, M1G, M5 and M5G were easily detected, in comparison with standards, in an interference-free area of the electropherogram. Diastereomeric separation of TR glucuronides in in vitro samples was achieved with 10 mM ammonium acetate-100 mM formic acid electrolyte solution at pH 2.75 and with basic micellar 25 mM tetraborate-70 mM SDS electrolyte solution at pH 10.45. Both separations showed that glucuronidation in vitro produces glucuronide diastereomers in different amounts. The authentic TR urine sample was also analysed by micellar method, but unambiguous identification of the glucuronide diastereomers was not achieved owing to many interferences.     

Automated trace enrichment for screening and/or determination of primary, secondary and tertiary amphetamines in biological samples by liquid chromatography

A rapid and simple liquid chromatographic method for the automated determination of amphetamines in biological fluids was developed. The proposed procedure is based on the injection of 250 microL of sample into a 20 x 2.1 mm id precolumn (packed with a 30 microns Hypersil C18 stationary phase) for enrichment and purification of the analytes. Next, the analytes are transferred to a 5 microns LiChrospher 100 RP18, 125 x 4 mm id analytical column for their separation under reversed-phase conditions. Water was used to eliminate the matrix components from the precolumn and a 0.2 M phosphate buffer (pH 3) containing 2% triethylamine was the mobile phase for the resolution of the amphetamines. The UV detector was set at 210 nm. The method was applied to the determination of different primary, secondary and tertiary amphetamines in plasma and urine: beta-phenylethylamine, norephedrine, ephedrine, N-methylpseudoephedrine, pseudoephedrine, N-methylephedrine, amphetamine, 3-phenylpropylamine and methamphetamine. The method provides satisfactory linearity and reproducibility within the tested concentration range (1.0-10.0 micrograms mL-1) and limits of detection of 50-500 ng/mL-1.