Direct High-performance Liquid Chromatography Method Research Articles

Simultaneous detection of residual diazepam, ketamine, nimetazepam, and xylazine by high-performance liquid chromatography: application in drug-spiked beverages for forensic investigation

ABSTRACT Drug facilitated crimes (DFCs) involve the incapacitation of a victim due to the administration of a single drug or a combination of drugs. Traditionally, biological samples are collected from a victim and analysed to provide evidence of drug administration. However, the rapid metabolism of many such drugs together with delays in analysis can compromise the detection of such substances. This study investigated the capacity of a high-performance liquid chromatography (HPLC) method to detect, simultaneously, four sedative-hypnotic drugs, diazepam, ketamine, nimetazepam, and xylazine, recovered from spiked beverages. A HPLC method was developed and validated and was then applied to the detection of the target substances in simulate forensic case scenarios. The four target substances were well-separated (correlation coefficient, 0.99) while the limit of detection was determined to be 39.1 ng/mL (xylazine), 78.1 ng/mL (diazepam and nimetazepam), and 156.3 ng/mL (ketamine), respectively. Precision (%RSD <7.8%) and accuracy (95.3%–106.6% recovery) were also acceptable and good recoveries were achieved from the drug-spiked samples. In conclusion, a simple and direct HPLC method was successfully developed and applied to the recovery of sedative-hypnotic drugs from spiked beverages.

Rapid direct analysis of retinyl palmitate (vitamin A) in fortified vegetable oils by HPLC-FLD

ABSTRACT Large-scale food fortification of vegetable oils with vitamin A has been implemented successfully for decades in numerous African and Asian countries, contributing demonstrably to reductions in vitamin A deficiency. For these programmes, reliable and validated analytical data are essential to demonstrate compliance with legal standards and fortification levels. Commonly, many analytical laboratories use a saponification method for the quantitative analysis of retinyl palmitate (the mostly used form of vitamin A for fortification) in fortified oils, which implies a multiple-step procedure with long analysis times and the potential risk of analyte loss. The aim of the present study was to develop and validate a direct High-performance Liquid Chromatography (HPLC) method that reduces these sample preparation steps, leading to the cost- and time-efficient quantification of retinyl palmitate in fortified oils. Oil samples are dissolved into the HPLC solvents, then injected directly into a common C18 column, and subsequently detected by a fluorescence detector. The limit of quantification (1.0 mg retinyl palmitate kg−1) and the working range of 1.0–100 mg retinyl palmitate kg−1 with a linearity of R2 = 0.9989 are appropriate to analyse fortified oil samples. The method also showed adequate precision (RSD between 1.1% and 3.1%) and recoveries (86–103%) at two different concentration levels. The accuracy of the direct HPLC method was additionally proven by the comparison of spiked samples with two external laboratories that used the saponification method. The robustness of the method was confirmed by the analysis of various spiked edible oils. The HPLC column is not deteriorated by the lipid matrix and shows excellent stability and long lifetime. Also, 9-cis-retinyl palmitate formed mainly by light exposure could be detected by this method. The direct HPLC method is a well-suited alternative to the saponification method for the rapid and reliable routine analysis of fortified oil samples.

Direct high-performance liquid chromatography method with refractometric detection designed for stability studies of treosulfan and its biologically active epoxy-transformers

Treosulfan (TREO) is an alkylating agent registered for treatment of advanced platin-resistant ovarian carcinoma. Nowadays, TREO is increasingly applied iv in high doses as a promising myeloablative agent with low organ toxicity in children. Under physiological conditions it undergoes pH-dependent transformation into epoxy-transformers (S,S-EBDM and S,S-DEB). The mechanism of this reaction is generally known, but not its kinetic details. In order to investigate kinetics of TREO transformation, HPLC method with refractometric detection for simultaneous determination of the three analytes in one analytical run has been developed for the first time.The samples containing TREO, S,S-EBDM, S,S-DEB and acetaminophen (internal standard) were directly injected onto the reversed phase column. To assure stability of the analytes and obtain their complete resolution, mobile phase composed of acetate buffer pH 4.5 and acetonitrile was applied. The linear range of the calibration curves of TREO, S,S-EBDM and S,S-DEB spanned concentrations of 20–6000, 34–8600 and 50–6000μM, respectively. Intra- and interday precision and accuracy of the developed method fulfilled analytical criteria. The stability of the analytes in experimental samples was also established. The validated HPLC method was successfully applied to the investigation of the kinetics of TREO activation to S,S-EBDM and S,S-DEB. At pH 7.4 and 37°C the transformation of TREO followed first-order kinetics with a half-life 1.5h.

Determination of octopamine and tyramine traces in dietary supplements and phytoextracts by high performance liquid chromatography after derivatization with 2,5-dimethyl-1H-pyrrole-3,4-dicarbaldehyde

The use of 2,5-dimethyl-1H-pyrrole-3,4-dicarbaldehyde (DPD) as a pre-column derivatization reagent for HPLC (high performance liquid chromatography) analysis of octopamine (oct) and tyramine (tyr) is proposed. The compound reacts under mild conditions (2 min at ambient temperature) with primary amino groups. The derivatization conditions were optimized by considering different parameters (temperature, time and reagent concentration). The synthesized oct and tyr adducts were characterized by (1)H NMR (nuclear magnetic resonance), ESI-MS (electrospray ionization mass spectrometry), IR (infrared) and UV (ultraviolet). Derivative chromatographic separations were performed on a Sinergy Hydro-RP column (150 mm × 4.6 mm i.d.) using a mobile phase consisting of methanol and triethylammonium phosphate buffer (pH 3; 10mM) at varying composition gradient elution and at a flow rate of 0.8 mL/min. Detection was set at λ=320 nm. The obtained results were compared with those achieved by a validated direct HPLC method with detection at λ=275 nm using a Sinergy Polar-RP column (250 mm × 3 mm i.d.) by isocratic elution conditions with a mobile phase consisting of methanol/acetonitrile/sodium pentanesulphonate (SPS; pH 3; 10mM), 7.5:7.5:85 (v/v/v) at a flow rate of 0.3 mL/min. Derivatization method sensitivity proved to be ten times higher than direct method. Limit of detection of oct and tyr was 0.010 and 0.008 μg/mL, respectively. The reliability of the pre-column method was satisfactory also in terms of linearity (from 0.028 to 1.255 and 0.024 to 1.244 μg/mL for oct and tyr, respectively), precision (relative standard deviation ≤2, without significant differences between intra-day and inter-day data) and recovery (from 98.9 to 101.2%). The proposed method showed to be suitable for a reliable determination of oct and tyr traces in commercially available phytoproducts using the instrumentation usually present in any analytical laboratory.

Direct separation and detection of biogenic amines by ion-pair liquid chromatography with chemiluminescent nitrogen detector

Analysis of biogenic amines is critical to pharmaceutical and food industry due to their biological importance. For many years, the determination of biogenic amines has relied on high performance liquid chromatography (HPLC) coupling with pre-, on-, or post-column derivatization procedures to enable UV or fluorescent detections. In this study, 14 biogenic amines were separated on a Phenomenex Luna ® Phenyl-Hexyl column by an ion-pair liquid chromatography method using perfluorocarboxylic acids as ion-pair reagents and detected by a chemiluminescent nitrogen detector (CLND). This direct separation and detection HPLC method eliminated the time consuming and cumbersome derivatization procedures. Compared with HPLC–UV (post-column derivatization with ninhydrin) and HPLC-charged aerosol detector (CAD) methods, this HPLC–CLND technique provided narrower peaks, better baselines, and improved separations and detections. Excellent linearity was acquired by CLND for each of the 14 biogenic amines ranging from less than 1 ng to about 1000 ng (on-column weights). The relative response factors determined by this LC–CLND method were proportional to the numbers of nitrogen atoms in each compound, which has been the characteristic of the equimolar determinations by CLND. In addition, a number of samples including beer, dairy beverage, herb tea, and vinegar were analyzed by the LC–CLND method with satisfactory precision and accuracy.

A direct HPLC method for the resolution and quantitation of the R-(−)- and S-(+)-enantiomers of vigabatrin (γ-vinyl-GABA) in pharmaceutical dosage forms using teicoplanin aglycone chiral stationary phase

A direct chiral high-performance liquid chromatography (HPLC) method was developed and validated for the resolution and quantification of antiepileptic drug enantiomers, R-(−)- and S-(+)-vigabatrin (gamma-vinyl-gamma-aminobutyric acid) in pharmaceutical products. The separation was optimized on a macrocyclic glycopeptide antibiotic chiral stationary phase (CSP) based on teicoplanin aglycone, chirobiotic (TAG), using a mobile phase system containing ethanol–water (80:20, v/v), at a flow rate of 0.4 ml/min and UV detection set at 210 nm. The stability of vigabatrin enantiomers under different degrees of temperature was also studied. The enantiomers of vigabatrin were separated from each other. The calibration curves were linear over a range of 100–1600 μg/ml ( r = 0.999) for both enantiomers. The overall recoveries of R-(−)- and S-(+)-vigabatrin enantiomers from pharmaceutical products were in the range of 98.3–99.8% with %RSD ranged from 0.48 to 0.52%. The limit of quantification (LOQ) and limit of detection (LOD) for each enantiomer were 100 and 25 μg/ml, respectively. No interferences were found from commonly co-formulated excipients.

Determination of alkyl nitrate additives in diesel fuel by liquid chromatography with infrared spectrometric detection

High-performance liquid chromatography (HPLC) using a variable wavelength infrared (IR) detector provides very selective monitoring of a particular functional group. A direct injection HPLC method was developed for the determination of amyl nitrate, hexyl nitrate, and octyl nitrate cetane improver additives in diesel fuel. The method was carried out for D-2 reference fuel spiked individually with three different levels of each additive. Recoveries were good except for low levels of octyl nitrate. Limits of detection for the method were estimated for peaks with a S/N ratio of 2. For amyl nitrate and hexyl nitrate, this was about 12 ..mu..g injected or 0.05 vol % in diesel fuel. Mixtures of all three additives can be determined with this method. It represents a time saving alternative to the wef chemical method for alkyl nitrates in diesel fuels. 1 figure, 3 tables.