Determination of the enantiomeric composition of chiral carboxylic acids using chiral derivatization and HPLC

Summary The enantiomers of chiral carboxylic acids were separated as their diastereomeric amides with (1R,2R)-(−)-1-(4-nitrophenyl)-2-amino-1,3-propanediol (“levobase”) and with “dextrobase” (the enantiomer of levobase) by high-performance liquid chromatography using a conventional C-18 column and various solvent systems containing acetonitrile, methanol, water, and phosphoric acid.     

HPLC enantioseparation of amino acids and their protected derivatives used in peptide synthesis with pre-column chiral derivatization

Summary Indirect chiral separation methods based on enantiomeric derivatizations were developed in order to monitor optical purity of uncoded amino acids and a new series of amino acid derivatives. Marfey's reagent was used for derivatization of amino groups: whilst boxyl groups were derivatised with (1R, 2R)-or (1S, 2S)-2-amino-1(4-nitrophenyl)-1,3-propanediol reagents were used, respectively. The separations of diastereomeric derivatives formed via derivatization were optimized in RP-HPLC and NP-HPLC systems. Presented at Balaton Symposium on High Performance Separation Methods, Siófok, Hungary, September 1–3, 1999     

Determination of paracetamol (acetaminophen) by HPLC with post-column enzymatic derivatization and fluorescence detection

Summary A novel method is described for the determination of paracetamol (acetaminophen;N-acetyl-4-aminophenol) in urine. After reversed-phase HPLC separation, paracetamol is oxidized by H₂O₂ with horseradish peroxidase catalysis. Detection is performed fluorimetrically at an excitation wavelength of 329 nm and an emission wavelength of 435 nm. Urine samples were spiked with paracetamol, diluted, and injected directly without further pretreatment. Under these conditions, the limit of detection was 2×10⁻⁸ molL⁻¹, and the limit of quantification was 7×10⁻⁸ molL⁻¹. The method was validated by two different approaches based on HPLC with UV-Vis detection.     

Direct determination of enantiomeric purity of FMOC amino acids with high performance liquid chromatography

Summary Analytical methods are described which allow a direct determination of enantiomeric purity of seventeen FMOC amino acids commonly used in peptide synthesis. The corresponding ester derivatives can be separated directly on cellulose tris(3,5-dimethylphenylcarbamate) (Chiralcel-OD). The methods are suitable for primary as well as secondary FMOC amino acids. The presence of a highly sensitive fluorescence moiety within the molecule, in combination with large separation factors (-values between 1.5–2.2) allowed for a general detection limit below 0.05%. In several cases the antipode has been determined in the ppm-range. An interesting result has been observed with respect to the elution order of the FMOC amino acid esters. The elution order of the Trp enantiomers is opposite to that obtained with the other amino acids. This is contrary to the generally held belief that elution order is identical within a homologous series of racemates when chromatographed under identical conditions on the same chiral stationary phase. In addition, the inversion of elution of the Pro enantiomers depending on the estertype indicates a competition of different separation mechanisms.     

HPLC determination of colistin and aminoglycoside antibiotics in feeds by post-column derivatization and fluorescence detection

Summary Reversed-phase, HPLC methods employing post-column derivatization and fluorescence detection were developed for the determination of the peptide colistin and four aminoglycoside antibiotics in feeds. Extraction of the analytes was by sonication and shaking with dilute hydrochloric acid. Post-column derivatization was performed using orthophtaldialdehyde-2-mercaptoethanol chemistry. Assay of colistin was by using an acetonitrile-aqueous sodium sulphate-triethylammonium phosphate (pH 2.8) eluent. Aminoglycoside antibiotics:amikacin, kanamycin, gentamycin and neomycin were analyzed using a tetrahydrofuran-aqueous sodium sulphate-sodium pentanesulphonate-acetic acid mobile phase. The method was also applied to some pharmaceutical preparations. Preliminary results showed that the method can be adapted for the assay of the above antibiotics in meat and animal serum for residue and pharmacokinetic studies. Presented at: Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, September 3–5, 1997.     

Direct enantiomeric analysis of amphetamine in plasma by simultaneous solid phase extraction and chiral derivatization

Summary An analytical approach has been developed for the one step determination of enantiomeric amphetamine composition in plasma, using on-line, pre-column solid phase derivatization with reversed phase HPLC separation. The high molecular weight protein components were excluded by the small pore structure of the polymer and washed out of the reaction column before derivatization. Spiked amphetamine in human plasma was extracted and derivatized by the polystyrene based FMOC-L-prolyl solid phase reagent. The derivatized diastereomers were separated on a conventional ODS column with an ACN/H₂O mobile phase. No kinetic resolution or racemization was observed in this solid phase derivatization. Calibration plots and reproducibility experiments were performed to demonstrate the validity of the new approach. Automation of the procedure provided a simple and reproducible method for direct chiral recognition in plasma samples.     

A post-column extraction system for the determination of tertiary amines by liquid chromatography with chemiluminescence detection

Summary The combination of an ion-pair extraction detection system with peroxyoxalate chemiluminescence detection has been investigated. Ion-pairs of protonated tertiary amines with a chemiluminescent counter ion are on-line post-column extracted to 1,2-dichloroethane containing bis(2,4,6-trichlorophenyl)oxalate (TCPO). Hydrogen peroxide is added to the organic phase by means of a solid-state perhydrit reactor. The influence of the base catalyst (imidazole) on the chemiluminescence reaction in apolar solvents was studied. Some sulfonated chemiluminophores were compared with respect to their chemiluminescence, ion-pairing and extraction properties, and 5-dimethylaminonaphthalene-1-sulfonate was found to be the most suitable reagent. The detection limit of the potential drug secoverine is in the sub-nanogram range.     

Optimisation of post-column reaction detector for HPLC of explosives

Summary Improvements in selectivity and sensitivity in the analysis of common explosives, like nitrate esters, nitramines and nitroaromatic compounds can be achieved by post-column derivatisation in a two step reaction detector. The first step in derivatisation is the photolysis of the analytes with UV at 254 nm. The photo reactor consists of a crocheted 20 m Tefzel capillary, which is coiled around a low pressure mercury lamp. In second step the nitrite ion generated is subsequently detected by a colourimetric reaction. The azo dye formed can be selectively detected at 540 nm.Addition of alkali after chromatographic separation to prevent oxidation of initially formed nitrite to nitrate during photolysis leads to a complex multistage arrangement. However, the contribution to peak broadening by the reactor is negligible and it is possible to detect 25–50 ppb of nitramines and 30–100 ppb of nitrate esters. Another advantage of the method is the selective detection of nitro compounds, even in complex matrices.The trace analysis of explosives is of growing interest in forensic science as well as in environmental analysis. It has been shown [1] that explosives can easily be extracted from soil and debris by the use of supercritical carbon dioxide. The separation and determination of explosives by gas chromatography is hindered by their thermal instability. In HPLC only the nitro aromatic explosives can be detected with sufficient sensitivity. Other types of explosives like the esters of nitric acid or nitramines do not absorb sufficiently in the UV region for sensitive detection. It has been shown [2] that explosives are liable to photochemical decomposition in the UV region, resulting in nitrate and nitrite, which have been detected after separation by ion-pair chromatography with electrochemical detection. A more sensitive and selective detection of nitrite has been possible in flow injection analysis [3]. Here a modified Griess reaction has been used. In a first step nitrite ions are used to form the diazonium salt with sulfanilamide which is coupled in a second step with N-[naphthyl-(1)]-ethylene diamine (NED) to form a redviolet azo dye with an absorption maximum at 540 nm. The advantage of this method is selective detection in the visible region, where hardly any other organic components are detected, which might be present in a crude environmental sample.In this paper the transfer of the Griess reaction to post-column derivatisation in RP chromatography of explosives will be described, and the optimisation of trace analysis of these solutes will be discussed.     

Analysis of basic hair dyes by HPLC with on-line post-column photochemical derivatisation

Summary A reversed phase liquid chromatographic method is proposed for the analysis of basic hair dyes (raw materials and colourant formulations). The performance of the method was enhanced by introducing post-column on-line photochemical derivatisation in combination with a Diode Array Detector. On-line photoderivatisation provided an effective way of selectively transforming the analytes to compounds with different spectral properties. For each analyte two characteristic UV-Visible spectra (photoreactor on and off) were obtained with the same mobile phase and this information in combination with the chromatographic data (k' at pH 3.0 and 4.5) enabled the unambiguous identification of both commonly used, approved, and banned basic hair dyes. Additionally, this approach was found useful to improve the method sensitivity, allowing the determination of analytes present in low concentration (0.03%) in complex commercial formulations.This work constitutes part of the thesis for the Dottorato di Ricerche of Roberto Gotti.     

Optimization of enantiomeric separations with chiral bonded phases

Summary The preparation and properties of chiral bonded phases with amino acid groups are described. These phases were used in non-polar eluents and in aqueous systems in the presence of Cu²⁺ ions in the ligand-exchange mode. The optimization of chiral resolution is demonstrated for both cases. With non-polar eluents similarity between bonded groups and solutes is required. The elution could be accelerated by non-protonic moderators. Ligand-exchange separation is influenced by the copper content of the eluent and the stationary phase, the organic moderator concentration, the pH and ionic strength of the buffer and by the temperature. Structural requirements of both the bonded groups and of the solutes for chiral separation are discussed.     

Trace analysis of sugars by HPLC and post-column derivatization

Summary A HPLC system with post-column derivatization for the quantitative analysis of sugars in complex matrices is described. As reagent a 0.2% solution of thymol in concentrated sulfuric acid has been used. The reaction is sensitive with reducing as well as non reducing sugars whereas sugar alcohols are discriminated. With this reagent and separation of sugars at high pH values with an anion exchange column it is possible to detect sugars in the ng range. Hence, it is possible to characterize wines not only by their fructose and glucose content but also by differences in the distribution of the other not fermentable sugars like trehalose, arabinose, galacturonic and glucuronic acid.     

Evaluation of novel dendrimer chiral stationary phases using HPLC

Summary The reversed phase chromatographic properties of the [G1]-L-glutamic and ethyl ester-AC-silica (1), [G2]-L-glutamic acid ethyl ester-AC-silica (2) and the [G1]-L-glutamic acidt-butyl ester-AC-silica (3) dendrimer stationary phases were evaluated. Initial studies involved the comparison between these phases with a classic reversed phase (i.e. ODS1) by the separation of a standard reversed phase test mixture composed of dimethylphthalate, nitrobenzene, anisole, diphenylamine and fluorene. Separations were achieved with comparable performance to those obtained with the conventional reversed phase (ODS1). However, it was apparent that the chromatographic selectivity exhibited by the dendrimer stationary phases was different from that of the ODS1 phase. On a per mole basis, the dendrimers exhibited similar (and sometimes greater) affinity for these analytes compared with the ODS1 ligand. Subsequent chromatographic experiments were conducted upon the dendrimer chiral stationary phases using chiral analytes under reversed phase and normal phase conditions. Chiral resolution was not observed.     

The on-line determination of the enantiomeric purity of enzymatically produced chiral compounds

Summary A prototype of a coupled-column chromatographic system has been developed to determine the enantiomeric purity of enzymatically produced chiral compounds. In this system, an immobilized enzyme HPLC reactor based upon -chymotrypsin (ACHT-IMER) was linked through a switching valve to a chromatographic column containing a crown ether-based chiral stationary phase (CR-CSP). Although ACHT is normally stereospecific for the L-enantiomorphs of aromatic amino acids, L-tryptophan (L-TRP), L-tyrosine and L-phenylalanine, we were able to achieve the hydrolysis of D-TRP methyl and ethyl esters on the ACHT-IMER. The percent of hydrolysis of the D-TRP esters could be manipulated by varying the resident time in the ACHT-IMER. In a series of experiments, D,L-TRP methyl or ethyl esters were injected into the ACHT-IMER, the flow stopped and after resuming the flow, the eluent which contained the hydrolyzed aminoacids, D- and L-TRP, was switched onto the CR-CSP where the enantiomeric composition of the TRP peak was determined. This configuration is a model for other IMER/CSP coupled-column systems which can be used for analytical and preparative applications.     

Development and validation of a chiral capillary electrophoresis method for assay and enantiomeric purity control of pramipexole

A rapid method for the enantioseparation of pramipexole and its R‐enantiomer has been developed by capillary electrophoresis. The influence of chemical and instrumental parameters was investigated including the type and concentration of chiral selectors, buffer composition and pH, co‐ions, applied voltage, capillary length and temperature. Optimal separation conditions were obtained using a 50 mM phosphate buffer (pH 2.8) containing 25 mM carboxymethyl‐β‐cyclodextrin on a fused‐silica capillary. Online UV detection was performed at 262 nm. A voltage of 25 kV was applied, and the capillary temperature was kept at 25°C. Hydrodynamic injection was performed at 3.45 kPa for 5.0 s. The separation of enantiomers was achieved in <6.5 min. The method was further validated in terms of stability of solutions, selectivity, linearity (both pramipexole and R‐enantiomer, R²>0.995), LOD and LOQ (0.91 and 2.94 μg/mL, respectively), repeatability (RSD<1.5%) and accuracy (pramipexole, 100.4%; R‐enantiomer, 100.5%). The proposed method was then applied to two kinds of pramipexole dihydrochloride monohydrate commercially available tablets, immediate release tablets (1.50 and 0.125 mg) and sustained release tablets (0.52 mg), to quantify the main component in the tablets. The amount of distomer could be quantified in bulk sample materials.     

柱前衍生化-HPLC测定仲丁胺对映体的纯度

用3, 5-二硝基苯甲酸对仲丁胺进行柱前衍生, 将其衍生物在CHIRALCEL OD-H手性固定相上拆分, 并通过二极管阵列紫外检测器对其衍生物进行检测, 建立了一种拆分仲丁胺消旋体、测定仲丁胺光学纯度的新方法. 以正己烷和乙醇或异丙醇为流动相, 在CHIRALCEL OD-H手性固定相上对仲丁胺衍生物进行了拆分, 并考察了流动相组成和柱温对该对映体衍生物分离的影响, 获得较好的分析条件, 分离因子大于1.2. 非手性试剂柱前衍生化法测定仲丁胺光学纯度与旋光度方法比较, 结果一致. 方法可用于仲丁胺对映体的质量控制.     

Enantioselective liquid-solid extraction for screening of structurally related chiral stationary phases

Summary An enantioselective liquid-solid batch extraction method is described for the screening of novel chiral stationary phases (CSPs) during optimization studies of chiral selectors derived form a common lead structure. Extraction enantioselectivity (α) values can be calculated from the enantiomeric excess ee-values of the selectand, which are measured in the liquid phase by enantioselective HPLC. Extraction α-values have been correlated with chromatographic α-values. The influence was studied of several experimental parameters of the assay (pH_a, buffer concentration, temperature, selector/selectand and phase ratio) on the enantiomeric excess (ee) values of the selectands and the enantioselectivity of the CSPs, respectively. The derived statistically significant model has then been implemented to predict chromatographic α-values of novel CSPs. For example, an ee of 89.3% for DNB-Leu as selectand could be achieved in batch extraction for a novel synthesized but mechanistically similarly-acting CSP derived form quinine. This corresponds to a calculated extraction α-value of 17.7. Based on this α_extraction a chromatographic α-value of 28.8 was predicted by the linear correlation model; the experimental HPLC α-value of 31.7 was in good agreement and demonstrated the validity of the proposed screening method. The method is particularly helpful in SO optimization studies.     

Direct enantiomeric separation of racemic precursors to diltiazem hydrochloride and clentiazem maleate by HPLC on a chiral ovomucoid column

Summary A direct HPLC separation method was developed for the determination of the enantiomers of racemic precursors to diltiazem (I) and its 8-chloro derivatives (II). The enantiomers were successfully separated on a chiral ovomucoid column using an aqueous-organic mobile phase (reversed-phase HPLC). The influence of the organic modifier and buffer pH on the retention and enantioselectivity was investigated. The chromatographic conditions chosen for the separation permitted complete resolution of the enantiomers of both the acid (I_b and II_b) and methyl ester precursors (I_a and II_a) within 20 min. The influence of sample load on retention times, theoretical plates numbers, peak heights and peak areas was also investigated. The peak areas showed a good linearity over the concentration range examined, although all the others were influenced significantly by the sample size. An optical antipode of the intermediate to be determined could be detected by the area-percentage method down to ca. 0.1%, together with the determination of its precursor, including its optical purity.     

Direct separation of the enantiomers of cetirizine and related compounds by reversed-phase chiral HPLC

Summary Direct separations of the enantiomers of cetirizine and related compounds have been achieved by reversed-phase HPLC on the Chiralcel OD-R, a polysaccharide-derived chiral stationary phase; the mobile phase was usually perchlorate solution supplemented with acetonitrile. Resolution of the enantiomers of cetirizine and related compounds was good. The effect of the acetonitrile content of the mobile phase was investigated, and the effect of the structure of the chiral compounds on their behavior on the Chiralcel OD-R column is discussed.     

Rapid screening of airway secretions for fucose by parallel ligand-exchange chromatography with post-column derivatization and fluorescence detection

Summary Fucose (6-deoxygalactose) is a constituent of airway mucous glycoproteins. In this paper we describe a high-throughput method for screening nasal lavage fluid samples and induced sputum samples for fucose. Fucose was released by hydrolysis with 0.5m sulfuric acid at 100°C for 4 h. After pH adjustment remaining proteins were removed by on-line dialysis. Chromatography was performed with two 300 mm×7.8 mm i.d. Bio-Rad Aminex HPX-87H columns arranged in a box-car configuration. Post-column derivatization was performed with benzamidine under alkaline conditions. Fluorescence was monitored at an excitation wavelength of 360 nm, using an optical cut-off filter of 420 nm. The limit of quantitation for fucose was 40 μm (S/N=3) in 300μL nasal lavage medium, with use of a 20-μL injection loop. Relative standard deviation (RSD) values for intra and inter assay data were below 15% and 20%, respectively, at spike levels of 635 μm l-fucose. The method was used to monitor the fucose content of human airway secretions. Presented at: 23^rd International Symposium on Chromatography, London, UK, October 1–5, 2000     

Characterization of peak purity by fast multiscan circular dichroism detection

Summary A method has been developed to detect inhomogeneity of apparently homogeneous peaks of very similar analytes. The method utilizes the rapid scan feature of state-of-the-art spectrometers/detectors that allow the recording of up to 30 spectra in a single chromatographic peak. Sensitivity and selectivity are enhanced by chiroptical/optical detection. Thus, identification of “front” and “rear” components of the peak can be carried out. The method is exemplified by mixtures of codeine, hydrocodone and oxycodone as analytes. Presented at: Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, September 3–5, 1997