SPE of 5‐lipoxygenase metabolites and the effect of head‐column field‐amplified sample stacking in MEKC

The SPE of leukotrienes and eicosatetraenoic acids using anion exchange materials was compared to the classical extraction with C₁₈ columns. A silica‐based strong anion exchanger, a polymer‐based weak anion exchanger, and a polymer‐based mixed‐mode strong anion exchanger were studied. All anion exchange materials displayed a higher recovery of the analytes with values between 70 and 90% when extracting standard solutions and analyzing by HPLC. The effect was less pronounced for the analysis of the compounds in incubations of polymorphonuclear leukocytes. Using MEKC with head‐column field‐amplified sample stacking for analyte quantification, much lower values of the peak areas were observed compared to the determination of the recovery of the analytes by HPLC. Using MEKC analysis, the highest values were found for the polymer‐based weak anion exchange material, while values below 10% were found for the polymer‐based mixed mode strong anion exchanger. This could be attributed to the presence of electrolytes in the eluates that compromised the stacking efficiency. The extent of residual electrolytes depended on the SPE protocol, resulting in large differences of the amount of analyte determined by MEKC when applying head‐column field‐amplified sample stacking for online analyte concentration.     

Macrocyclic polyamine‐functionalized silica as a solid‐phase extraction material coupled with ionic liquid dispersive liquid–liquid extraction for the enrichment of polycyclic aromatic hydrocarbons

In this study, silica modified with a 30‐membered macrocyclic polyamine was synthesized and first used as an adsorbent material in SPE. The SPE was further combined with ionic liquid (IL) dispersive liquid–liquid microextraction (DLLME). Five polycyclic aromatic hydrocarbons were employed as model analytes to evaluate the extraction procedure and were determined by HPLC combined with UV/Vis detection. Acetone was used as the elution solvent in SPE as well as the dispersive solvent in DLLME. The enrichment of analytes was achieved using the 1,3‐dibutylimidazolium bis[(trifluoromethyl)sulfonyl]imide IL/acetone/water system. Experimental conditions for the overall macrocycle‐SPE–IL‐DLLME method, such as the amount of adsorbent, sample solution volume, sample solution pH, type of elution solvent as well as addition of salt, were studied and optimized. The developed method could be successfully applied to the analysis of four real water samples. The macrocyclic polyamine offered higher extraction efficiency for analytes compared with commercially available C₁₈ cartridge, and the developed method provided higher enrichment factors (2768–5409) for model analytes compared with the single DLLME. Good linearity with the correlation coefficients ranging from 0.9983 to 0.9999 and LODs as low as 0.002 μg/L were obtained in the proposed method.     

Toxicological screening of human plasma by on‐line SPE‐HPLC‐DAD: identification and quantification of basic drugs and metabolites

An automated multi‐analyte screening method for the identification and quantification of 92 drugs and metabolites based on on‐line solid‐phase extraction–high‐performance liquid chromatography–diode array detection technique was developed and successfully validated. In addition, a database with 870 entries including UV‐spectra, relative/retention times and response factors of toxicologically relevant compounds was created. Plasma samples (0.2 mL) were treated with methanol, diluted with buffer and on‐line extracted (Strata X, 20 ×2 mm, 25 µm) at pH 9. Analytical separation was carried out on a Gemini NX column (150 ×4.6 mm, 3 µm) using gradient elution with acetonitrile–water (90:10,v/v) and 0.05 m potassium dihydrogen phosphate buffer (pH 2.3). Linear calibration curves with correlation coefficients ≥0.9950 were obtained for 78 analytes. As an additional benefit, the newly developed method allows the quantification of 42 analytes (e.g. antidepressants, neuroleptics and anticonvulsants) in a concentration range suitable for therapeutic drug monitoring. Limits of quantitation ranged from 0.02 mg/L (chlordiazepoxide) to 3.4 mg/L (mexiletine). Inter‐ and intra‐day precisions of quality control samples (low/high) were better than 15% (zolpidem) and accuracy (bias) ranged from ?11% (opipramol, venlafaxine) to 11% (venlafaxine, trazodone). Tests for carry‐over and sample stability under different storage conditions were also performed and stability was adequate. Four cases of poisoning analysis are presented. Copyright © 2014 John Wiley & Sons, Ltd.     

HPLC‐ESI‐MS/MS analysis and pharmacokinetics of luteoloside,a potential anticarcinogenic component isolated from Lonicera japonica,in beagle dogs

Luteoloside is a potential anticarcinogenic component isolated from Lonicera japonica, a traditional Chinese medicine (TCM). This study details the development and validation of a sensitive and accurate HPLC‐ESI‐MS/MS method for the quantification of luteoloside in dog plasma. Sample pretreatment includes simple protein precipitation using methanol–acetonitrile (1:1, v/v). A Phenomenex Gemini C₁₈ column (2.0 × 50 mm, i.d., 3.5 µm) was used to separate luteoloside and internal standard by gradient mode with mobile phase consisting of water containing 0.1% formic acid and methanol containing 0.1% formic acid at a flow rate of 0.40 mL/min with a column temperature of 25°C. The detection was performed by positive ion electrospray ionization (ESI) in multiple reaction monitoring mode. The calibration curves were linear (R > 0.995) over the concentration range 1.0–2000 ng/mL and the lower limit of quantification was 1.0 ng/mL. The intra‐day and inter‐day precisions (RSD) were all <15%, accuracies (RE) were within the range of ±15%, and recoveries were between 85.0 and 115%. The validated HPLC‐ESI‐MS/MS method was successfully applied to determine plasma concentrations of luteoloside after intravenous administration of luteoloside at a dose level of 20 mg/kg. Copyright © 2012 John Wiley & Sons, Ltd.     

Determination of Carbon‐based Engineered Nanoparticles in Marketed Fish by Microwave‐assisted Extraction and Liquid Chromatography‐atmospheric Pressure Photoionization‐tandem Mass Spectrometry

An optimized method for the determination of two major carbon‐based engineered nanoparticles (C₆₀ and C₇₀) in marketed fish samples is described. The method involves the use of microwave‐assisted extraction (MAE) coupled with liquid chromatography ‐ tandem mass spectrometry with atmospheric pressure photoionization (LC‐APPI‐MS/MS). Factors affecting the extraction efficiency of the analytes from fish samples were optimized by a central composite design method. The optimal extraction temperature and time for MAE were found to be 233 °C for 22 min, and the extraction solution was composed of toluene and acetone in a ratio of 4.64:1. The limits of quantitation (LOQs) were 0.1 and 0.05 ng/g for C₆₀ and C₇₀, respectively. The precision for these analytes at two spiked levels, as indicated by relative standard deviations (RSDs), were less than 10% for both intra‐ and inter‐day analysis. Accuracy, expressed as the mean extraction recovery, was between 85 and 98%. The method was further validated based on EU Commission Decision 2002/657/EC, including a decision limit (CCα) and detection capability (CCβ) for marketed fish samples.     

Development and validation of an HPLC‐UV method for the simultaneous determination of the antipsychotics clozapine,olanzapine and quetiapine,several beta‐blockers and their metabolites

A simple, accurate and selective column‐switching high‐performance liquid chromatography (HPLC) method was developed and validated for simultaneous quantification of six beta ‐blockers (metoprolol, timolol, bisoprolol, propranolol, carvedilol and nebivolol), three of their metabolites (α ‐hydroxy metoprolol, N‐ desisopropyl propranolol and 4′‐hydroxy carvedilol 4‐HCAR), three antipsychotics (olanzapine, clozapine and quetiapine) and three of their metabolites (N‐ desmethyl olanzapine, N‐ desmethyl clozapine and N‐ desalkyl quetiapine) in human serum. After pretreatment on a Merck LiChrospher RP‐4 ADS column (25 μm), drugs were separated on a Phenomenex Gemini Phenyl Hexyl 110 A column (250 × 4.6 mm, 5 μm) using a gradient mixture of acetonitrile and potassium dihydrogen phosphate buffer pH 3.1 (containing 10% methanol) as a mobile phase at a flow rate of 1 mL/min. The total analysis time was 40 min. For detection of the analytes, four different UV wavelengths were used: 215, 226, 242 and 299 nm. The method was validated according to the guidelines of the Society of Toxicology and Forensic Chemistry in terms of selectivity, linearity, accuracy, precision and stability and successfully applied for the analysis of the 15 described analytes in human serum.     

Comparison of C18 silica and multi‐walled carbon nanotubes as the adsorbents for the solid‐phase extraction of Chlorpyrifos and Phosalone in water samples using HPLC

A comparison between C₁₈ silica and multi‐walled carbon nanotubes (MWCNTs) in the extraction of Chlorpyrifos and Phosalone in environmental water samples was carried out using HPLC. Parameters affecting the extraction were type and volume of elution solvent, pH and flow rate of sample through the adsorbent. The optimum conditions obtained by C₁₈ cartridge for adsorption of these pesticides were 4 mL dichloromethane as elution solvent, sample pH of 5, flow rate of 1 mL/min, and those for MWCNT cartridge were 3 mL dichloromethane, pH of 5 and flow rate of 10 mL/min, respectively. Optimized mobile phase for separation and determination of these compounds by HPLC was methanol/water (80:20 v/v) with pH=5 (adjusted with phosphate buffer). Under optimal chromatographic and SPE conditions, LOD, linear range and precision (RSD n=8) were 3.03×10^?3, 0.01–5.00 μg/mL and 2.7% for Chlorpyrifos and 4.03×10^?4, 0.01–5.00 μg/mL and 2.3% for Phosalone, in C₁₈ cartridge, respectively. These values for MWCNT were 4.02×10^?6, 0.001–0.500 μg/mL and 1.8% for Chlorpyrifos and 1.02×10^?6, 0.001–0.500 μg/mL and 1.5% for Phosalone, respectively.     

Simultaneous determination of quinoxaline‐1,4‐dioxides in feeds using molecularly imprinted solid‐phase extraction coupled with HPLC

A simple, selective, and reproducible molecularly imprinted SPE coupled with HPLC method was developed for monitoring quinoxaline‐1,4‐dioxides in feeds. Molecularly imprinted polymers were synthesized in methanol using mequindox (MEQ) as template molecule and acrylamide as functional monomer by bulk polymerization. Under the optimum SPE conditions, the novel polymer sorbents can selectively extract and enrich carbadox, MEQ, quinocetone, and cyadox from a variety of feeds. The molecularly imprinted SPE cartridge was better than nonimprinted, C₁₈, and HLB cartridges in terms of both recovery and precision. Mean recoveries of four quinoxaline‐1,4‐dioxides from six kinds of feeds spiked at 1.0, 10, and 100 mg/kg ranged between 75.2 and 94.7% with RSDs of less than 10%. The decision limits (CCαs) and the detection capabilities (CCβs) of four analytes were 0.15–0.20 mg/kg and 0.44–0.56 mg/kg, respectively. The class selectivity of the polymers was evaluated by checking three drugs with different molecular structures to that of MEQ.     

Simultaneous determination of acetylsalicylic acid and salicylic acid in human plasma by isocratic high‐pressure liquid chromatography with post‐column hydrolysis and fluorescence detection

A selective, sensitive and rapid high‐performance liquid chromatography method with post‐column hydrolysis and fluorescence detection was developed for the simultaneous quantification of acetylsalicylic acid and its metabolite salicylic acid in human plasma. Following the addition of 2‐hydroxy‐3‐methoxybenzoic acid as internal standard and simple protein precipitation with acetonitrile, the analytes were separated on a ProntoSIL 120 C₁₈ ace‐EPS column (150 × 2 mm, 3 µm) protected by a C₈ guard column (5 µm). The mobile phase, 10 mm formic acid in water (pH 2.9) and acetonitrile (70:30, v/v), was used at a flow rate of 0.35 mL/min. After on‐line post‐column hydrolysis of acetylsalicylic acid (ASA) to salicylic acid (SA) by addition of alkaline solution, the analytes were measured at 290 nm (λ_ex) and 400 nm (λ_em). The method was linear in the concentration ranges between 0.05 and 20 ng/μL for both ASA and SA with a lower limit of quantification of 25 pg/μL for SA and 50 pg/μL for ASA. The limit of detection was 15 pg/μL for SA and 32.5 pg/μL for ASA. The analysis of ASA and SA can be carried out within 8 min; therefore this method is suitable for measuring plasma concentrations of salicylates in clinical routine. Copyright © 2012 John Wiley & Sons, Ltd.     

Development and validation of an LC‐MS/MS method for quantification of Δ9‐tetrahydrocannabinolic acid A (THCA‐A), THC,CBN and CBD in hair

For analysis of hair samples derived from a pilot study (‘in vivo’ contamination of hair by sidestream marijuana smoke), an LC‐MS/MS method was developed and validated for the simultaneous quantification of Δ9‐tetrahydrocannabinolic acid A (THCA‐A), Δ9‐tetrahydrocannabinol (THC), cannabinol (CBN) and cannabidiol (CBD). Hair samples were extracted in methanol for 4 h under occasional shaking at room temperature, after adding THC‐D₃, CBN‐D₃, CBD‐D₃ and THCA‐A‐D₃ as an in‐house synthesized internal standard. The analytes were separated by gradient elution on a Luna C18 column using 0.1% HCOOH and ACN + 0.1% HCOOH. Data acquisition was performed on a QTrap 4000 in electrospray ionization‐multi reaction monitoring mode. Validation was carried out according to the guidelines of the German Society of Toxicological and Forensic Chemistry (GTFCh). Limit of detection and lower limit of quantification were 2.5 pg/mg for THCA‐A and 20 pg/mg for THC, CBN and CBD. A linear calibration model was applicable for all analytes over a range of 2.5 pg/mg or 20 pg/mg to 1000 pg/mg, using a weighting factor 1/x. Selectivity was shown for 12 blank hair samples from different sources. Accuracy and precision data were within the required limits for all analytes (bias between ?0.2% and 6.4%, RSD between 3.7% and 11.5%). The dried hair extracts were stable over a time period of one to five days in the dark at room temperature. Processed sample stability (maximum decrease of analyte peak area below 25%) was considerably enhanced by adding 0.25% lecithin (w/v) in ACN + 0.1% HCOOH for reconstitution. Extraction efficiency for CBD was generally very low using methanol extraction. Hence, for effective extraction of CBD alkaline hydrolysis is recommended. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of polycyclic aromatic hydrocarbons in water by a novel mesoporous‐coated stainless steel wire microextraction combined with HPLC

A novel mesoporous‐coated stainless steel wire microextraction coupled with the HPLC procedure for quantification of four polycyclic aromatic hydrocarbons in water has been developed, based on the sorption of target analytes on a selectively adsorptive fiber and subsequent desorption of analytes directly into HPLC. Phenyl‐functionalized mesoporous materials (Ph‐SBA‐15) were synthesized and coated on the surfaces of a stainless steel wire. Due to the high porosity and large surface area of the Ph‐SBA‐15, high extraction efficiency is expected. The influence of various parameters on polycyclic aromatic hydrocarbons extraction efficiency were thoroughly studied and optimized (such as the extraction temperature, the extraction time, the desorption time, the stirring rate and the ionic strength of samples). The results showed that each compound for the analysis of real water samples was tested under optimal conditions with the linearity ranging from 1.02×10^?3 to 200 μg/ L and the detection limits were found from 0.32 to 2.44 ng/ L, respectively. The RSD of the new method was smaller than 4.10%.     

Simultaneous determination of multiveterinary drug residues in pork meat by liquid chromatography‐tandem mass spectrometry combined with solid phase extraction

An LC‐MS/MS method developed for simultaneous analysis of 54 veterinary drug residues of six families in pork meat samples, including sulfanilamide, nitroimidazoles, quinolones, macrolide antibiotics, lincosamides, and praziquantel. The pork meat sample was prepared by extraction with ACN, and clean‐up on a C₁₈ SPE cartridge. The sample was separated on a C₈ column and eluted with ACN, methanol, and formic acid. The MS/MS detector is operated in the multiple reaction monitoring mode, acquiring two specific precursor‐product ion transitions per target compound. The method showed excellent linearity (R² ≥ 0.99) and high precision (relative SD, RSD ≤ 19.8%) for all compounds. The method quantification limits of 54 veterinary drug residues were in the range of 0.3–3.0 μg/kg. Recoveries for most analytes based on matrix‐matched calibration in matrices were 20.9–121.0%. This method has been successfully applied for analysis of more than 100 pork meat samples from the local market; five of the 54 drugs were detected.     

Rapid high‐performance liquid chromatography–tandem mass spectrometry method for simultaneous measurement of venlafaxine and O‐desmethylvenlafaxine in human plasma and its application in comparative bioavailability study

A rapid high‐performance liquid chromatography–tandem mass spectrometry method has been developed and validated for simultaneous measurement of venlafaxine and O‐desmethylvenlafaxine in human plasma using fluoxetine as an internal standard. In the liquid–liquid extraction method, compounds and internal standard were extracted from plasma using methyl tertiary butyl ether as an extraction solvent. The HPLC separation of the analytes was performed on a Zorbax SB‐C₁₈, 50 × 4.6 mm, 5 µm column, using a isocratic elution program using a mobile phase consisting of HPLC‐grade methanol: 5 mm ammonium acetate (80:20 v/v) at a flow‐rate of 1.0 mL/min with a total runtime of 3.0 min. The proposed method has been validated with a linear range of 4–400 ng/mL for venlafaxine and 5–500 ng/mL for O‐desmethyl venlafaxine. The method was applied for a bio‐equivalence study of 75 mg tablets formulation in 32 Indian male healthy subjects under fasting conditions. Copyright © 2009 John Wiley & Sons, Ltd.     

A rapid and simple RP‐HPLC method for quantification of kirenol in rat plasma after oral administration and its application to pharmacokinetic study

A rapid and simple reverse‐phase high‐performance liquid chromatography (RP‐HPLC) was developed and validated for the quantification of kirenol in rat plasma after oral administration. Kirenol and darutoside (internal standard, IS) were extracted from rat plasma using Cleanert™ C₁₈ solid‐phase extraction (SPE) cartridge. Analysis of the extraction was performed on a Thermo ODS‐2 Hypersil C₁₈ reversed‐phase column with a gradient eluent composed of acetonitrile and 0.1% phosphoric acid. The flow rate was 1.0 mL/min and the detection wavelength was set at 215 nm. The calibration curve was linear over the range of 9.756–133.333 µg/mL (r² = 0.9991) in rat plasma. The lower limits of detection and quantification were 2.857 and 9.756 µg/mL, respectively. The intra‐ and inter‐day precisions (relative standard deviation, RSD) were between 2.24 and 4.46%, with accuracies ranging from 91.80 to 102.74%. The extraction recovery ranged from 98.16 to 107.62% with RSD less than 4.81%. Stability studies showed that kirenol was stable in preparation and analytical process. The present method was successfully applied to the pharmacokinetic study of kirenol in male Sprague–Dawley rats after oral administration at a dose of 50 mg/kg. Copyright © 2010 John Wiley & Sons, Ltd.     

Fast microwave‐assisted extraction of rotenone for its quantification in seeds of yam bean (Pachyrhizus sp.)

The aim of this study was to find if fast microwave‐assisted extraction could be an alternative to the conventional Soxhlet extraction for the quantification of rotenone in yam bean seeds by SPE and HPLC‐UV. For this purpose, an experimental design was used to determine the optimal conditions of the microwave extraction. Then the values of the quantification on three accessions from two different species of yam bean seeds were compared using the two different kinds of extraction. A microwave extraction of 11 min at 55°C using methanol/dichloromethane (50:50) allowed rotenone extraction either equivalently or more efficiently than the 8‐h‐Soxhlet extraction method and was less sensitive to moisture content. The selectivity, precision, trueness, accuracy, and limit of quantification of the method with microwave extraction were also demonstrated.     

Dicationic imidazolium ionic liquid modified silica as a novel reversed‐phase/anion‐exchange mixed‐mode stationary phase for high‐performance liquid chromatography

A dicationic imidazolium ionic liquid modified silica stationary phase was prepared and evaluated by reversed‐phase/anion‐exchange mixed‐mode chromatography. Model compounds (polycyclic aromatic hydrocarbons and anilines) were separated well on the column by reversed‐phase chromatography; inorganic anions (bromate, bromide, nitrate, iodide, and thiocyanate), and organic anions (p‐aminobenzoic acid, p‐anilinesulfonic acid, sodium benzoate, pathalic acid, and salicylic acid) were also separated individually by anion‐exchange chromatography. Based on the multiple sites of the stationary phase, the column could separate 14 solutes containing the above series of analytes in one run. The dicationic imidazolium ionic liquid modified silica can interact with hydrophobic analytes by the hydrophobic C₆ chain; it can enhance selectivity to aromatic compounds by imidazolium groups; and it also provided anion‐exchange and electrostatic interactions with ionic solutes. Compared with a monocationic ionic liquid functionalized stationary phase, the new stationary phase represented enhanced selectivity owing to more interaction sites.     

On‐line flow‐through extraction–preconcentration‐large volume injection‐RP LC for trace determination of pyrethroids in Slovak soil

A rapid micro‐analytical multiresidue method was developed for analysis of pyrethroids (kadethrin K, cypermethrin C and permethrin P) in soil micro‐sample (200 mg). It uses on‐line flow‐through extraction of soil micro‐samples (packed into a short glass column) with a methanol‐aqueous citric acid buffer mixture, successive on‐line SPE preconcentration of analytes from the extract and on‐line RP‐HPLC analysis with UV photometric detection. The separation of pyrethroids is performed on a Purospher RP‐18e column with methanol/water as mobile phase. Effects of sorbent placed at the bottom of a short column holding the soil sample and different kinds of on‐line SPE columns were tested. Besides, the influence of volume of the effluent on the pyrethroids recovery was also studied. Calibration curves were linear over the range assayed from 0.01 to 0.2 μg/mL with correlation coefficients of linear regression (least‐squares method) in the range 0.998–0.999. Recovery studies were carried out at 0.25–1.00 μg/g dry soil fortification level and obtained recoveries were for K 81–84%, C 56–59% and for P 58–63%. Achieved LOD (confidence band) of studied pyrethroids were for large‐volume injection (1 mL) 4.5 ng K, 3.7 ng C, 3.6 ng P or 27 ng/g K, 32 ng/g C and 29 ng/g P in dry soil “solid sampling HPLC”.     

Quantitative HPLC method and pharmacokinetic studies of ergosta‐4,6,8(14),22‐tetraen‐3‐one,a natural product with diuretic activity from Polyporus umbellatus

A simple and specific HPLC method with dual wavelength UV detection for the determination of ergosta‐4,6,8(14),22‐tetraen‐3‐one (ergone) in rat plasma was developed and proved to be efficient. The method used ergosterol as internal standard (IS). Following a single‐step protein precipitation, the analyte and IS were separated on an Inertsil ODS‐3 column with a mobile phase containing methanol–water (99:1, v/v) at a flow rate of 1 mL/min. The analytes were detected by using UV detection at wavelength of 350 (ergone) and 283 (IS) nm, respectively. The calibration curve was linear over the range of 0.1–2.0 µg/mL and the lower limit of quantification was 0.1 µg/mL. The intra‐day and inter‐day precision studies showed good reproducibility with RSD less than 8.5%. The intra‐day and inter‐day accuracy ranged from 95.6 to 104%. Mean extraction recovery was above 95% at the low, medium and high concentrations. The present HPLC‐UV method was simple and reliable. The method described herein had been successfully applied for the pharmacokinetic studies in male SD rats after administration of 20 mg/kg dose of solution of ergone. Copyright © 2010 John Wiley & Sons, Ltd.     

Separation of cannabinoids on three different mixed‐mode columns containing carbon/nanodiamond/amine‐polymer superficially porous particles

Three mixed‐mode high‐performance liquid chromatography columns packed with superficially porous carbon/nanodiamond/amine‐polymer particles were used to separate mixtures of cannabinoids. Columns evaluated included: (i) reversed phase (C₁₈), weak anion exchange, 4.6 × 33 mm, 3.6 μm, and 4.6 × 100 mm, 3.6 μm, (ii) reversed phase, strong anion exchange (quaternary amine), 4.6×33 mm, 3.6 μm, and (iii) hydrophilic interaction liquid chromatography, 4.6 × 150 mm, 3.6 μm. Different selectivities were achieved under various mobile phase and stationary phase conditions. Efficiencies and peak capacities were as high as 54 000 N/m and 56, respectively. The reversed phase mixed‐mode column (C₁₈) retained tetrahydrocannabinolic acid strongly under acidic conditions and weakly under basic conditions. Tetrahydrocannabinolic acid was retained strongly on the reversed phase, strong anion exchange mixed‐mode column under basic polar organic mobile phase conditions. The hydrophilic interaction liquid chromatography column retained polar cannabinoids better than the (more) neutral ones under basic conditions. A longer reversed phase (C₁₈) mixed‐mode column (4.6 × 100 mm) showed better resolution for analytes (and a contaminant) than a shorter column. Fast separations were achieved in less than 5 min and sometimes 2 min. A real world sample (bubble hash extract) was also analyzed by gradient elution.