Mobile Phase Additives Research Articles

Effect of Ionic Liquids as the Mobile Phase Additives on the HPLC Resolution of Four Active Compounds from Sophora flavescens Ait

The retention behaviour of four active compounds from Sophora Flavescens Ait using three ionic liquids as mobile phase modifiers was examined. The effect of the pH and the amount of ionic liquid modifier on the retention of these compounds was determined in methanol/water (v/v) as the mobile phase containing different ionic liquids ranging in concentration from 0.1 mmol/L to 3.0 mmol/L. The ionic liquids showed promise as additives in high-performance liquid chromatography.

Ionic liquids as mobile phase additives in high-performance liquid chromatography with electrochemical detection: Application to the determination of heterocyclic aromatic amines in meat-based infant foods

The beneficial effects of several ionic liquids (ILs) as mobile phase additives in high-performance liquid chromatography with electrochemical detection for the determination of six heterocyclic aromatic amines (HAs) have been evaluated for first-time. The studied ionic liquids were 1-butyl-3-methylimidazolium tetrafluoroborate (BMIm-BF4), 1-hexyl-3-methylimidazolium tetrafluoroborate (HMIm-BF4) and 1-methyl-3-octylimidazolium tetrafluoroborate (MOIm-BF4). Several chromatographic parameters have been evaluated in the presence or absence of ILs, or using ammonium acetate as the most common mobile phase additive, with three different C18 stationary phases. The effect of the acetonitrile content was also addressed. In general, best resolution, lower peak-widths (up to 72.1% lower) and lower retention factors are obtained when using ILs rather than ammonium acetate as mobile phase additives. The main improvement was obtained in the baseline noise, being 360% less noisy for BMIm-BF4, 310% for HMIm-BF4, and 227% for MOIm-BF4, when compared to ammonium acetate at +1000mV. Different chromatographic methods using the best conditions for each IL were also evaluated and compared. Finally, the best chromatographic conditions using 1mM of BMIm-BF4 as mobile phase additive, the Nova-Pak® C18 column, 19% (v/v) of acetonitrile content in the mobile phase, and +1000mV in the ECD, have been applied for the chromatographic analysis of six HAs contained in meat-based infant foods. The whole extraction method of meat-based infant foods using focused microwave-assisted extraction and solid-phase extraction has also been optimized. Extraction efficiencies up to 89% and detection limits ranged between 9.30 and 0.165ngg−1 have been obtained under optimized conditions.

Effect of Mobile Phase pH and Organic Content on LC--MS Analysis of Nucleoside and Nucleotide HIV Reverse Transcriptase Inhibitors

The HIV-1 reverse transcriptase inhibitors tenofovir (TFV), emtricitabine (FTC), and lamivudine (3TC) are widely used in the treatment of HIV-1-infected persons and are now being considered as chemoprophylactic drugs for the prevention of sexual HIV transmission. Assays that measure these drugs after either oral or topical application are critical to the understanding of the pharmacokinetic profiles of the drugs and allow a rational design of chemoprophylaxis modalities for evaluation in macaque models and human trials. We developed a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) method for sensitive measurement of FTC, 3TC, and TFV in plasma from macaques. To achieve detection limits of 10 pg on column, the plasma analytes were measured using acidic mobile phase and positive electrospray ionization MS-MS detection. However, this caused various chromatographic peak distortions, which were minimized by using mobile phase additives that induced ion-pairing interactions. Chromatographic peak tailing was minimized by adjusting the organic mobile phase concentration while considering the simultaneous effect of organic content on buffer and analyte pKa. Injection solution interferences were corrected by chromatographic peak focusing using column switching. The final method provides simultaneous measurement of all three analytes with a wide linear range of 1-3000 ng/mL using 0.1 mL plasma (10 pg on column) and coefficients of variation from 5% to 15% in the high ng/mL concentration range and from 16% to 20% in the low ng/mL concentration range.

Effects of Ionic Liquid on the Separation of 2-Chlorophenol and 2,4,6-Trichlorophenol in RP-HPLC

In this study, the use of ionic liquids as additives in the separation of 2-chlorophenol and 2,4,6-trichlorophenol in reversed-phase high-performance liquid chromatography is reported. The ionic liquids used were 1-butyl-3-methylimidazolium tetrafluoroborate, 1-ethyl-3-methylimidazolium tetrafluoroborate, 1-ethyl-3-methylimidazolium methylsulfate ([EMIm][MS]), and 1-octyl-3-methylimidazolium methylsulfate ([OMIm][MS]), because of their high hydrophobicity and good solvating properties. The effects of the ionic liquid on retention factor and resolution, and the change of detector response using the different ionic liquids were studied. Separation with the [MS] anion ionic liquid in the eluent resulted in better resolution than with the [BF4] anion ionic liquid. The best result was achieved using 3.0 mM/L of [OMIm][MS] and 1.0 mM/L [EMIm][MS] as mobile phase additives.

Enantiomeric separation of phenylsuccinic acid by cyclodextrin-modified reversed phase high-performance liquid chromatography

The chiral separation of phenylsuccinic acid (PSA) was studied by reversed phase high-performance liquid chromatography (RP-HPLC) with cyclodextrins (CDs) as chiral mobile phase additives. The effects of types of CDs, concentration of hydroxypropyl-β-cyclodextrin (HP-β-CD), percentage of organic modifier, pH value and column temperature on enantioselective separation were investigated. The quantification property of the developed RP-HPLC method was examined. The chiral recognition mechanism of PSA was also discussed. The results show that a baseline separation of PSA enantiomers is achieved on a Lichrospher C18 column (4.6 mm (inner diameter)×250 mm, 5 μm) with HP-β-CD as chiral mobile phase additive. The capacity factors of R-PSA and S-PSA are 3.94 and 4.80, respectively. The separation factor and resolution are respectively 1.22 and 8.03. The mobile phase is a mixture of acetonitrile and deionized water (20:80, volume ratio) containing 10 mmol/L HP-β-CD and 0.05% trifluoroacetic acid (pH 2.5, adjusted with triethylamine) with a flow rate of 1.0 mL/min. The ultraviolet (UV) detector is set at 254 nm. The likely roles are inclusion interaction, induction and hydrogen bonding between HP-β-CD and PSA enantiomers.

Rapid method for the quantitative determination of efavirenz in human plasma

A pharmacokinetic interaction study between efavirenz (EFV), a non-nucleoside reverse transcriptase inhibitor used in the treatment of HIV-1 infection, and an African traditional medicine, African potato in human subjects was undertaken. This necessitated the development and validation of a quantitative method for the analysis of EFV in plasma. A simple mobile phase consisting of 0.1M formic acid, acetonitrile and methanol (43:52:5) was pumped at a low flow rate of 0.3ml/min through a reverse phase Phenomenex® Luna C18 (2) (5μm, 150mm×2.0mm i.d.) column maintained at 40°C. Diclofenac sodium was used as an internal standard (IS) and EFV and IS were monitored at 247nm and 275nm, respectively. A simple and rapid sample preparation involved the addition of mobile phase to 100μl of plasma to precipitate plasma proteins followed by direct injection of 10μl of supernatant onto the column. The procedures were validated according to international standards with good reproducibility and linear response (r=0.9990). The intra- and inter-day accuracies were between 12.3 and 17.7% at the LLOQ and between −5.8 and 9.1% for the QC samples. The intra- and inter-day precision of EFV determinations were 5.1 or less and 7.2% RSD or less, respectively across the entire QC concentration range. Mean recovery based on high, medium and low quality control standards ranged between 92.7 and 94.1% with %RSD values better than 3%. Plasma samples were evaluated for short-term (ambient temperature for 6h) and long-term (−10±2°C for 60 days) storage conditions and were found to be stable. The method described is cost-effective and has the necessary accuracy and precision for the rapid quantitative determination of EFV in human plasma.

Improvement of Chiral Discrimination of Acidic Enantiomers on Teicoplanin Stationary Phase by the Use of Chaotropic Effect

This work reveals new advantages of using inorganic salts with chaotropic anions (perchlorate, hexafluorophosphate) as mobile phase additives in enantioseparation of acidic compounds (mandelic acid, tropic acid, phenyllactic acid, 2-(p-methoxyphenoxy)-propionic acid) on teicoplanin-based stationary phase in reversed phase mode. It was observed that the retention factors (k) and the separation factors (α) of acidic analytes increased in the presence of salts in the mobile phase. The standard changes in enthalpy (ΔH0), entropy (ΔS0), and the free energy (at 20°C) (ΔG0) of the binding interactions between enantiomers and the chiral selector in the stationary phase were calculated from the experimental relationships of ln k vs 1/T in the presence of chaotropic salts in eluent systems. The Van't Hoff plots reveal that the interaction mechanism in the presence of chaotropic additives is enthalpy driven. Enthalpy-entropy compensation (EEC) studies revealed mechanistic similarity in retention of the investigated compounds.

Enantioseparation of cetirizine by chromatographic methods and discrimination by 1H‐NMR

Cetirizine is an antihistaminic drug used to prevent and treat allergic conditions. It is currently marketed as a racemate. The H1-antagonist activity of cetirizine is primarily due to (R)-levocetirizine. This has led to the introduction of (R)-levocetirizine into clinical practice, and the chiral switching is expected to be more selective and safer. The present work represents three methods for the analysis and chiral discrimination of cetirizine. The first method was based on the enantioseparation of cetirizine on silica gel TLC plates using different chiral selectors as mobile phase additives. The mobile phase enabling successful resolution was acetonitrile-water 17: 3, (v/v) containing 1 mM of chiral selector, namely hydroxypropyl-beta-cyclodextrin, chondroitin sulphate or vancomycin hydrochloride. The second method was a validated high performance liquid chromatography (HPLC), based on stereoselective separation of cetirizine and quantitative determination of its eutomer (R)-levocetirizine on a monolithic C18 column using hydroxypropyl-beta-cyclodextrin as a chiral mobile phase additive. The resolved peaks of (R)-levocetirizine and (S)-dextrocetirizine were confirmed by further mass spectrometry. The third method used a (1)H-NMR technique to characterize cetirizine and (R)-levocetirizine. These methods are selective and accurate, and can be easily applied for chiral discrimination and determination of cetirizine in drug substance and drug product in quality control laboratory. Moreover, chiral purity testing of (R)-levocetirizine can also be monitored by the chromatographic methods.

Fluoroquinolone antibiotic determination in bovine, ovine and caprine milk using solid-phase extraction and high-performance liquid chromatography-fluorescence detection with ionic liquids as mobile phase additives

This paper describes the use of 1-ethyl-3-methylimidazolium tetrafluoroborate (EMIm-BF 4) as mobile phase additive for the analysis by high-performance liquid chromatography with fluorescence detection of a group of seven basic fluoroquinolone antibiotics (i.e. fleroxacin, ciprofloxacin, lomefloxacin, danofloxacin, enrofloxacin, sarafloxacin and difloxacin) in different milk samples. EMIm-BF 4 was found superior to 1-butyl-3-methylimidazolium tetrafluoroborate for the separation of the analytes from chromatographic interferences of the sample matrix. The optimized method was applied to the analysis of ovine, caprine and bovine milk, in the last case in either skimmed, semi-skimmed and full-cream milk after suitable acidic deproteination followed by a solid-phase extraction procedure. Recovery values between 73% and 113% were obtained for the three types of bovine milk samples, as well as for ovine and caprine milk (RSDs below 16% in all cases), which clearly demonstrates the applicability of the method to the three types of milk irrespective of the fat content of the samples. Limits of detection were in the range of 0.5–8.1 μg/L (approximately 0.5–25.9 μg/kg), well below the maximum residue limits established for these compounds by the current European legislation. A screening study of 24 different milk samples was also developed. In none of the samples, residues of the selected antibiotics were found.

Mobile phase additives for enhancing the chromatographic performance of astaxanthin on nonendcapped polymeric C30‐bonded stationary phases

Astaxanthin shows peak deformation and reduced peak area response when eluted with methanol and methyl tert-butyl ether on nonendcapped polymeric C30-bonded HPLC phases. The present study tested different column manufacturers, column batches, and ten mobile phase additives including acids, bases, buffers, complexing and antioxidant agents for improvement of peak shape and peak area response. Concerning chromatographic benefits and feasibility, ammonium acetate was found to be the best additive followed by triethylamine for all columns tested. Variation of the mobile phase pH equivalent and the column temperature showed no synergistic effects on peak shape and peak area response. Results indicate that peak tailing and variation of peak area response are due to different on-column effects. Possible mechanisms of the observed phenomenon will be discussed.

Separation of fudosteine on an achiral C18column by HPLC with chiral ligand-exchange selectors as mobile phase additives

Summary A reversed-phase chiral liquid chromatographic method had been developed and validated for resolution of the enantiomers of racemic fudosteine. The effects on the separation of the amounts of anhydrous cupric sulfate and l-phenylalanine, the methanol content, mobile phase pH, and temperature were investigated. The method was validated for linearity, repeatability, intermediate precision, sample recovery, solution stability, and limits of detection (LOD). l-Phenylalanine and anhydrous cupric sulfate as chiral ligand-exchange complexes were used for separation, isomer identification, related substance investigation, and analysis of fudosteine enantiomers in fudosteine bulk drugs and fudosteine tablets.

Determination of the lipophilicity of selected isoquinoline alkaloids by RP-TLC

Summary Nine alkaloids were chromatographed by reversed phase (RP) TLC on RP-18 with aqueous acetone or aqueous dioxane mobile phases. A variety of mobile phase additives (ammonia, diethylamine (DEA), tetrabutylammonium chloride (TBA-Cl)) were used to suppress ionization of the alkaloids and/or reduce ionic interactions with surface silanol groups. Ion-pair (IP) RP-TLC was also performed with aqueous acetone mobile phases and reagents such as pentane sulphonic acid (PSA), octane sulphonic acid (OSA), or di-(2-ethylhexyl)orthophosphoric acid (HDEHP) as mobile phase additives. In these systems retention of the alkaloids was determined using mobile phases with different modifier concentrations. Relationships between RM and modifier concentration were sought. A linear semilogarithmic equation was fitted to experimental data and used to obtain lipophilicity values RMW (RM for pure water), the slope (S), and ϕ0, the intercept with the x-axis. The retention of standards with known lipophilicity log P was then determined using the chromatographic systems described above and RMW values were calculated. From these experimental data equations relating log P and RMW were created for each system separately and these equations were used to estimate log Pexp values for the alkaloids. log Pexp, slope (S), and ϕ0 values obtained by use of different TLC systems were also correlated.

Validated Chiral LC Method for the Enantiomeric Separation of Palonosetron Hydrochloride

A new and accurate chiral liquid chromatographic method has been developed for the separation of palonosetron hydrochloride (PALO) and its (R,R)-enantiomer in bulk drug samples with an elution time of about 20 min. The chromatographic separation was carried out by normal phase chromatography using an immobilized cellulose based chiral stationary phase (Chiralpak-IC) with a mobile phase composed of n-hexane:ethanol:1,4 dioxane:trifluoroacetic acid:diethylamine (65:30:5:0.3:0.3, v/v) pumped at a flow rate of 1.0 mL min−1. The resolution (Rs) between the enantiomers was found to be greater than 3.0 and interestingly the (R,R)-enantiomer was eluted prior to the (S,S)-enantiomer (PALO) in the developed method. Mobile phase additives, trifluoroacetic acid and diethylamine played a key role in achieving chromatographic resolution between the enantiomers and also in enhancing chromatographic efficiency. The limit of detection (LOD) and limit of quantification (LOQ) of the (R,R)-enantiomer were found to be 0.03 and 0.1 μg respectively for 10 μL injection volume. The developed method shows excellent linearity (r2 > 0.999) over a range of LOQ to 0.3% for the (R,R)-enantiomer. The percentage recovery of the (R,R)-enantiomer in bulk drug samples ranged from 97.2 to 102.3 revealing good sensitivity of the developed method. Robustness studies were also carried out on the developed method.

Direct TLC Resolution of (±)-Ketamine and (±)-Lisinopril by Use of (+)-Tartaric Acid or (−)-Mandelic Acid as Impregnating Reagents or Mobile Phase Additives. Isolation of the Enantiomers

Direct resolution of the enantiomers of the racemic drugs ketamine and lisinopril has been achieved by TLC. Enantiomerically pure tartaric acid and mandelic acid were used as chiral impregnating reagents and as mobile phase additives. When (−)-mandelic acid was used as chiral impregnating reagent use of ethyl acetate–methanol–water 3:1:1 (v/v) as mobile phase enabled successful resolution of the enantiomers of both compounds. For lisinopril, the mobile phase acetonitrile–methanol–water–dichloromethane 7:1:1:0.5 (v/v) was successful when (+)-tartaric acid was used as impregnating agent. When (+)-tartaric acid was used as mobile phase additive the mobile phase acetonitrile–methanol–(+)-tartaric acid (0.5% in water, pH 5)–glacial acetic acid 7:1:1.1:0.7 (v/v) enabled successful resolution of the enantiomers of lisinopril. The effects on resolution of temperature, pH, and the amount of chiral selector were also studied. The separated enantiomers were isolated and identified. Spots were detected with iodine vapour. LODs were 0.25 and 0.27 μg for each enantiomer of ketamine with (+)-tartaric acid and (−)-mandelic acid, respectively, whereas for lisinopril LODs were 0.14 and 0.16 μg for each enantiomer with (+)-tartaric acid (both conditions) and (−)-mandelic acid, respectively.

Ionic liquids as mobile phase additives for the high-performance liquid chromatographic analysis of fluoroquinolone antibiotics in water samples

In this work, four ionic liquids differing in the length of the alkyl chain on the imidazolium cation and one ionic liquid containing tetraethylammonium, all with the same counterion, (i.e. 1-ethyl-3-methylimidazolium tetrafluoroborate (EMIm-BF(4)), 1-butyl-3-methylimidazolium tetrafluoroborate (BMIm-BF(4)), 1-hexyl-3-methylimidazolium tetrafluoroborate (HMIm-BF(4)), 1-methyl-3-octylimidazolium tetrafluoroborate (MOIm-BF(4)), and tetraethylammonium tetrafluroborate (Et(4)N-BF(4))) were tested as mobile phase additives for HPLC separation of a group of seven basic fluoroquinolone (FQ) antibiotics for human and veterinary use (i.e. fleroxacin, ciprofloxacin, lomefloxacin, danofloxacin, enrofloxacin, sarafloxacin, and difloxacin) using a conventional reversed-phase Nova-Pak C(18) column. Fluorescence detection was used. Among the ionic liquids selected, use of BMIm-BF(4) enabled effective separation of these compounds with relatively low analysis time (14 min). The best separation was achieved by isocratic elution at 1 mL min(-1) with 5 mmol L(-1) BMIm-BF(4) and 10 mmol L(-1) ammonium acetate at pH 3.0 with 13% (v/v) acetonitrile. Limits of detection (LODs) for fluorescence detection were in the range 0.5-11 microg L(-1). The method was tested by analyzing several water samples after the optimization of a suitable solid-phase extraction (SPE) procedure using Oasis HLB cartridges. Mean recovery values were above 84% for all analytes with LODs in the range 1-29 ng L(-1).

Enhancing Productivity in the Analytical Laboratory Through the Use of Ultra Fast-HPLC in Preformulation/Formulation Development

The pharmaceutical industry today is driven to create new, more efficient ways to discover, develop, deliver and monitor drugs. Pharmaceutical companies are being faced with major challenges in reducing drug discovery and development timelines. Automation and the introduction of new analytical technologies that increase speed of analysis are integral in the analytical laboratory. The development of rapid chromatographic methods in preformulation and formulation development is playing an increasing role to support this drive in efficiency and productivity. The introduction of ultra fast-HPLC systems that can operate at pressures of up to 15,000 psi with columns packed with sub-2-µm particles have allowed for high speed and efficient separations. The consequent reduction of time, solvent, and waste disposal, and the analysis of more samples per unit time makes ultra-fast HPLC a very attractive technology. Faster method development and decision making can be achieved during late-phase preformulation/formulation development for the analysis of both singly charged and multiply charged basic compound analysis. The use of ultra fast-HPLC (UHPLC) for pH scouting experiments and the determination of the analyte's ionogenic nature was shown to be an effective tool for rapid and systematic method development. The implementation of this technology was also evaluated for the analysis of drug product formulations and excipient compatibility studies. Increased speed of analysis and significant gains in resolution per unit time were obtained compared to separations performed using conventional HPLC systems (operating pressures of <5,500 psi). Also, the use of liophilic ions as mobile phase additives with operation under high pressures led to enhanced separation selectivity, retention, and peak symmetry of multiply charged basic compounds.

Improved efficiency in micellar liquid chromatography using triethylamine and 1-butanol as mobile phase additives to reduce surfactant adsorption

The effect of triethylamine as a mobile phase modifier on chromatographic efficiency in micellar liquid chromatography (MLC) is reported for nine different columns with various bonded stationary phases and silica pore sizes, including large-pore short alkyl chain, non-porous, and perfluorinated. Reduced plate height ( h) versus reduced velocity ( ν) plots were constructed for each column and the A′ and C′ terms calculated using a simplified Van Deemter equation introduced in our previous work. To further explore the practicality of using triethylamine in the micellar mobile phase, the efficiency of nine polar and non-polar substituted benzenes was studied on seven columns. Surfactant adsorption isotherms were measured for five columns with three micellar mobile phases to understand the relationship between adsorbed surfactant, mobile phase additive, and column efficiency. Clear improvements in efficiency were observed with the addition of 2% (v/v) triethylamine to a 1-butanol modified aqueous micellar mobile phase. This finding is supported by the lower amount of surfactant adsorbed onto the stationary phase when TEA is present in the mobile phase compared to an SDS only or a 1-butanol modified SDS mobile phase.

Influence of Inorganic Mobile Phase Additives on the Retention and Separation Efficiency of Selected Amino Acids in Thin-Layer Chromatography on Cellulose Layers

Selected amino acid standards are investigated on cellulose layers using organic-aqueous eluent systems modified with neutral and chaotropic salts: chlorides, iodides, nitrates, thiocyanates, perchlorates, and hexafluorophosphates at low concentrations ranging from 10 up to 80mM in whole mobile phase. The effect of salts used as the mobile phase modifier is analyzed by the comparison of densitograms, peak symmetry coefficient, and theoretical plate number. The efficiency of chromatographic systems modified with inorganic salts additives depends primarily on the kind of salt and organic solvent in the mobile phase. The best efficiency is obtained through the addition of ammonium thiocyanate to the mobile phase containing acetonitrile as an organic modifier.

Liophilic Mobile Phase Additives in Reversed Phase HPLC

The separation of basic compounds can be challenging and the use of inorganic mobile phase additives have been successfully used in chromatographic methods development. About fifteen years ago the role of these additives as ion-interaction agents for selectively adjusting retention of ionic analytes was discovered and the theory of chaotropicity was applied to reversed phase chromatography. In the last ten years the studies of the influence of these counterions on the retention of ionizable analytes and the interaction with the stationary phase in various hydro-organic eluents has expanded our knowledge of this phenomenon. The general view and understanding of the process have been significantly updated and the use of these ionic additives (liophilic ions) in the mobile phase has become regular practice in the pharmaceutical industry for optimization and fine tuning of complex separations. This paper reviews the latest developments in the field and discusses the modification and expansion of our theoretical understanding of the process. The paper also describes their application in practical separations for a wide variety of analytes, from small molecules to peptides and even chiral separations.

Interaction of native α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin and their hydroxypropyl derivatives with selected organic low molecular mass compounds at elevated and subambient temperature under RP-HPLC conditions

The main focus of this study was to explore the capability of native alpha-cyclodextrin, beta-cyclodextrin and gamma-cyclodextrin and their hydroxypropyl derivatives for host-guest interaction with 7,8-dimethoxyflavone, selected steroids (estetrol, estriol, estradiol, estrone, testosterone, cortisone, hydrocortisone, progesterone and 17alpha-hydroxyprogesterone) and polycyclic aromatic hydrocarbons (toluene, naphthalene, 1,8-dimethylnaphthalene, 1-acenaphthenol, acenaphthylene and acenaphthene) under reversed-phase liquid-chromatography conditions. The study revealed that native cyclodextrins interact more efficiently with the analytes investigated than do their hydroxypropyl counterparts. In the low-temperature region, enormously high ratios were observed for polycyclic aromatic hydrocarbons, particularly 1,8-dimethylnaphthalene, acenaphthene and acenaphthylene chromatographed on a beta-cyclodextrin-modified mobile phase. In such a case, the retention times of the polycyclic aromatic hydrocarbons were strongly reduced (e.g. from 127 to 1.2 min for 1,8-dimethylnaphthalene) and were close to the hold-up time of the high-performance liquid chromatography (HPLC) system (0.7 min). Moreover, chiral separation of 1-acenaphthenol optical isomers was observed and the elution order of the enantiomers was determined. Within the steroids group, strong interaction was observed for estradiol and testosterone. The results of cluster analysis indicate that beta-cyclodextrin as well as gamma-cyclodextrin and its hydroxypropyl derivative can be most effective mobile-phase additives under reversed-phase HPLC conditions for 3D-shape-recognition-driven separation, performed at subambient and elevated temperatures, respectively.