Automated high-performance liquid chromatographic determination of amphetamine in biological fluids using column-switching and on-column derivatization

Summary A rapid and simple liquid-chromatographic method has been developed for on-line quantification of amphetamine in biological fluids. Untreated samples (20 μL) are injected directly into the chromatographic system and purified on a 20 mm×2.1 mm i.d. pre-column packed with 30 μm Hypersil C₁₈ stationary phase. After clean-up the analyte is transferred to the analytical column (125 mm×4 mm i.d., 5 μm LiChrospher 100 RP₁₈) for derivatization and separation using a mixture of acetonitrile and the derivatization reagent (o-phthaldialdehyde andN-acetyl-L-cysteine) as the mobile phase. The experimental conditions for on-line derivatization and resolution of the amphetamine have been optimized, and the results have been compared with those obtained by derivatizing the analyte in pre-column mode. The method described has been applied to the determination of amphetamine in plasma and urine. Good linearity and reproducibility were obtained in the 0.1–10.0 μg mL⁻¹ concentration range, and limits of detection were 25 ng mL⁻¹ and 10 ng mL⁻¹ with UV and fluorescence detection, respectively. The procedure described is very simple and rapid, because no off-line manipulation of the sample is required; the total analysis time is approximately 8 min.     

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.     

Automatic in-tube SPME and fast liquid chromatography: a cost-effective method for the estimation of dibuthyl and di-2-ethylhexyl phthalates in environmental water samples

A 80-cm length commercially available capillary coated with 95% polydimethylsiloxane and 5% polydiphenylsiloxane (TBR-5) was employed to carry out on-line extraction and preconcentration of dibuthyl phthalate (DBP) and di-2-ethylhexyl phthalate (DEHP) in the chromatographic system. The coated capillary was placed between the sample injection loop and the injection needle of an autosampler. Variables affecting the automatic in-tube solid-phase microextraction (SPME) were optimized. A Genesis C₁₈ (5 cm × 4.6 mm i.d., 4 μm particle size) was employed as analytical column. The achieved limits of detection by use of diode array detection were 1 and 2.5 μg L⁻¹, respectively. The proposed conditions have been applied to determine those compounds at low ppb levels (≤250 μg L⁻¹) in aqueous samples. No matrix effect was found, and recoveries between 85 and 115% were obtained. The precision of the method was good, and the achieved intra- and inter-day variation coefficients were between 5 and 20%. The analysis time per sample was 20 min and any off-line pre-treatment of the samples was needed. The taken sample volume was 100 μL. Data on the application of the described method to the analysis of different water samples are presented.     

A Guide to Avoid Method Bias of Chromium (III,VI) Chemiluminescence Determination by Luminol-Hydrogen Peroxide Reaction - Application to Water Samples

Cr(III) and/or Cr(VI) determinations based on light emission produced by luminol oxidation by hydrogen peroxide in basic aqueous solution catalyzed by Cr(III) were studied in order to diagnose and/or avoid method bias. The calibration step was optimized, and the usefulness of the method for speciating chromium was tested. The use of the standard addition method in the linear interval concentration range made it possible to diagnose the accuracy of the method for real samples. Good results were obtained for several real water samples containing chromium at different concentrations. The proposed protocol made the method traceable with an appropriate certified reference material and with the reference method.     

Separation of the enantiomers of primary and secondary amphetamines by liquid chromatography after derivatization with (−)-1-(9-fluorenyl)ethyl chloroformate

Summary The chiral reagent (−)-1-(9-fluorenyl)ethyl chloroformate (FLEC) has been evaluated for the enantioselective analysis of amphetamines by liquid chromatography. For separation of the FLEC diastereomers conventional reversed-phase conditions were used. The conditions affording the best enantiomeric resolution and sensitivity were determined for amphetamine, methamphetamine, ephedrine, pseudoephedrine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), and 3,4-methylenedioxyethylamphetamine (MDE). All the amphetamines assayed could be separated with resolution factors ranging from 0.91 to 1.92. Although FLEC is typically used as a fluorogenic reagent, it was shown that UV detection is the best option for stereospecific analysis of the methylenedioxylated amphetamine derivatives (MDA, MDMA, and MDE), because different fluorimetric responses were obtained for the corresponding diastereomers. The utility of the proposed conditions for stereoselective analysis of amphetamines in different types of sample is discussed.     

Elimination of the unknown irrelevant matrix absorbance by using the H-Point Standard Additions Method (HPSAM)

It is demonstrated how the H-Point Standard Additions Method (HPSAM) using DeltaA as analytical signal (from data at three previously selected wavelengths) is only related with analyte concentration when unknown irrelevant matrix absorbance is present. The method is compared with the most common previously reported methods, such as derivative spectroscopy or the compensation method. The obtained results show that the proposed HPSAM leads to the same found concentration of analyte as the other reported methods, except detection limits and standard deviation for six replicates which are lower, because of the use of absorbance data (instead of first derivative data as usual). In addition, the obtained data permit us to differentiate between linear and non-linear spectral behaviour for the interferent. The method was also applied to routine analyses using single-standard calibration.     

Kinetic and chemometric studies of the determination of creatinine using the Jaffé reaction. Part I. Kinetics of the reaction: analytical conclusions

A kinetic-spectrophotometric study of the Jaffé reaction was carried out and the kinetic behaviour, calibration step and interfering effect of albumin on creatinine standard solutions were studied. It was concluded that there is a variation in the kinetic behaviour of the system when higher concentrations of creatinine, picrate or sodium hydroxide are tested. The experimental conditions for quantifying creatinine must be chosen so that the kinetic behaviour is the same in the dynamic concentration range. Changes in the absorbance (delta A) versus concentration equations were chosen as the most suitable for calibration graphs. It was also shown that creatinine results will have a proportional bias error if the interfering effect of albumin is not taken into consideration.     

Derivatization of ephedrine with o-phthaldialdehyde for liquid chromatography after treatment with sodium hypochlorite

The usefulness of the reaction with NaClO followed by derivatization with o-phthaldialdehyde (OPA) and N-acetyl-L-cysteine (NAC) has been investigated for the chromatographic analysis of ephedrine. The influence of parameters affecting the two-stage reaction has been evaluated, including concentration of NaClO, time of reaction, temperature and pH. On the basis of these studies, conditions for the pre-column and (automated) post-column determination of ephedrine are presented. The described conditions have been applied to the measurement of ephedrine in the concentration intervals 0.2-20.0 microg/ml and 2.0-50.0 microg/ml for the pre-column and post-column methods, respectively. The possibility of applying the NaClO/ OPA-NAC method to other primary, secondary and tertiary derivative amphetamines has also been evaluated.     

H-point standard additions method for resolution of overlapped chromatographic peaks with a conventional fluorescence detector. Determination of phenol and cresols in waters

Summary The H-Point Standard Additions Method (HPSAM) is proposed in order to resolve overlapping peaks in liquid chromatography by using a conventional fluorescence detector. The method uses as analytical signals the heights or the areas obtained at two previously selected emission wavelengths, and good results are obtained for highly overlapping peaks with highly overlapping fluorescence spectra. The principal benefits of the method are the ease of finding the required wavelengths, its insensitivity to changes in the retention time of the peak from one injection to another, and the possibility of using it in highly or only partially overlapping peaks. We have applied the method to the determination of phenol and cresols in water, resolving by the proposed method the overlapping peaks ofm- andp-cresol.     

Influence of water sample storage protocols in chemiluminescence detection of trace elements

This paper shows the influence of different sample storage protocols, on the chemiluminescence signal of some metal ions. The storage protocols studied were: acid addition (HCl or HNO(3)) and no reagent addition to filtered and refrigerated (T=4 degrees C) samples. Light emission was produced for the chemiluminescence reaction between luminol and hydrogen peroxide in buffer carbonate conditions (pH 10.8) catalysed by Cr(III), Co(II) and Cu(II). Batch and/or flow modes in different conditions were tested. Fe(II), Fe(III), Ni(II) and Mn(II) did not give chemiluminescence in the studied conditions. A parallel study of sensitivity and selectivity was performed. Then the presence or absence of the masking agent EDTA, added to samples or used in the carrier stream, is assayed. If the samples are acidified with HNO(3), a previous neutralisation is needed using batch mode. The determination of Cr(III) is independent of storage protocol by flow injection (FI) method; however, the determination of Co(II) or Cu(II) or total determination of three metals requires the conditioning of standards. Detection limits achieved are ranged between 0.5 and 2 mug l(-1). For batch mode, detection limits are better for unacidified samples and worse for carbonate-neutralised samples. The influence of storage protocols was validated using standard metal mixtures and calibration solutions. The use of standard reference material (SRM(c) 1640) (Trace elements in natural water) corroborates the previous statements and validates the accuracy of the different approaches underlined. This paper demonstrates that it is possible to determine Cr(III) selectively in natural waters.