Development of a bimodal polarimetric response model for improved quantitation of enantiomeric mixtures under conditions of poor chromatographic resolution

The combination of a chiral-selective separation mode with polarimetric detection was investigated in terms of its ability to quantitate enantiomeric mixtures of the dansylated derivatives of phenylalanine, threonine, and valine under conditions of poor chromatographic resolution. Using the difference between two Gaussian functions to model the bimodal response obtained using polarimetric detection and incomplete chiral resolution, correlation coefficients as high as 0.999 were obtained when actual enantiomeric fractions were plotted against observed enantiomeric fractions calculated from the fitting procedure. The methodology was evaluated at different levels of chromatographic resolution, and as a function of the sign and magnitude of the specific rotation of the model compounds. Limits of quantitation calculated at different levels of enantiomeric excess demonstrate the potential of the method to quantitate enantiomeric mixtures even under conditions of poor chromatographic resolution. Minimum measurable quantities (MMQ) at, or below the 1.0% enantiomeric excess (ee) level were obtained for the three amino acid derivatives tested under various conditions of chromatographic resolution when the polarimetric data were fit to the bimodal response model. Even under the lowest resolution conditions tested, the MMQ was determined to be at the 5% ee level.     

Enantiomeric resolution of bupivacaine by high-performance liquid chromatography and chiroptical detection

This work reports a new analytical procedure for the separation and determination of the enantiomers of bupivacaine and the determination of the enantiomeric purity. The isomers were separated using a Chirex 3020 (250 mm x 4.6 mm) with a mobile phase of n-hexane:dichloroethane:ethanol (82:9:9, v/v/v) at a flow-rate of 1 ml min(-1) and UV, polarimetric and circular dichroism (CD) detection. Obtained retention times were 5.93 and 7.53 min (R and S) with a resolution of Rs=2.36. Precisions (RSD) were 1.83 and 2.02% (CD detection) and 3.07 and 1.26% (UV detection) for R- and S-enantiomers, respectively (at 10 microg level). Detection limits were 0.5 and 0.5 microg (R and S) with CD detection, and 0.9 and 0.3 microg with UV detection. Polarimetric detection was inadequate to perform a quantitative method at similar concentration ranges as UV and CD because of poor sensitivity. A procedure for determination of enantiomeric purity using a conventional chromatographic column (RP18, Luna) coupled to a CD detector and anisotropy factor (CD/UV) as analytical signal was also developed. Obtained results show that RSDs of 6.7-1.6% were obtained in the range of 0-100% enantiomeric purity.     

Improvements in the determination of penicillin analogues by HPLC separation and laser-based polarimetric detection

The combination of HPLC separation and laser-based polarimetric detection is shown to provide unique advantages when applied to the study of penicillin analogues. The mass delectability for penicillin G is 5 ng with this system, which is an order-of-magnitude improvement over other techniques. More importantly, the polarimetric system can provide specific rotation information about eluting species. Individual specific rotations are reported for the (10R)- and (10S)-epimers of both carbenicillin and ticarcillin. These results demonstrate the sensitivity of specific rotation to the arrangement of atoms at or near the chiral centre, suggesting that specific rotation may be used to identify closely related penicillin analogues.     

Improvements in the qualitative and quantitative study of erythromycins using laser-based polarimetric detection

A technique in which liquid chromatographic separation is combined with laser-based polarimetric detection is described for the study of erythromycin and its analogs. The polarimetric system, owing to the inherent selectivity of the technique, can provide unique advantages, particularly when the measurement must be made in a complicated matrix. Individual specific rotations have been measured for the three erythromycin analogs A, B and C and for the derivative erythromycin ethylsuccinate (EES). The results suggest that specific rotation may be used to identify closely related erythromycin species in studies of their fate and location in physiological pathways. The detection limit for the technique is 12 ng for erythromycin and 10 ng for EES. The distinct advantages of this technique, in terms of both sensitivity and selectivity, are demonstrated by the determination of erythromycin in milk and EES in a pharmaceutical preparation.     

HPLC separation of tetracycline analogues: comparison study of laser-based polarimetric detection with UV detection

A sensitive and specific high-performance liquid chromatography (HPLC) method based upon laser-based polarimetric detection is developed for the determination of six tetracycline analogues. By interfacing the laser-based polarimeter online with an HPLC system, the specific rotation of each analogue is obtained as compounds elute from the separation system. The six structurally similar tetracycline analogues exhibit significant differences in specific rotations. The experiments suggest that specific rotation can be useful in identifying closely related tetracycline analogues. Linear relationships are found to be in the range of 0.342-0.0043 mg for the tetracycline analogues. Five of the six analogues exhibit excellent linearity (R(2) value >/= 0.99). The polarimetric results are compared with UV detection. The HPLC-laser-based polarimetric detection instrument is able to quantitate the studied tetracycline analogues with high precision, accuracy, and sensitivity, which make it useful for the development of a standard method for the determination of tetracyclines in biological specimens. The performance of the HPLC-polarimetric system for the analysis of tetracyclines in a biological matrix is evaluated. The selectivity of polarimetric detection provides a distinct advantage in the analysis of tetracycline analogues in milk. The HPLC-polarimetric system provides a rapid and sensitive technique that involves minimal sample cleanup and pretreatment for the analysis of tetracyclines in milk.     

Determination of cisplatin and related platinum complexes in plasma ultrafiltrate and urine by high-performance liquid chromatography with on-line radioactivity detection

A reversed-phase ion-pair chromatographic method with on-line radioactivity detection for the simultaneous determination of 195mPt-labelled cisplatin and related platinum complexes has been developed. With this system a good resolution of various radiolabelled platinum complexes can be achieved. The detection limit of the radioactivity detector is 10 ng of cisplatin (specific activity of 15 MBq/mg cisplatin) per millilitre of urine or plasma ultrafiltrate. The detector response is independent of both the chemical structure of the platinum complexes and the matrix composition of the samples. This method may serve as a reference system for other high-performance liquid chromatographic systems with less specific and sensitive detectors.     

Voltammetric/amperometric detection for liquid chromatography

A voltammetric/amperometric detector based on a dual-electrode electrochemical detector is described for liquid chromatography. The detector combines the advantages of both voltammetric and amperometric detection. A three-dimensional data array of current response as a function of both time (chromatographic domain) and potential (electrochemical domain) is obtained. From the chromatographic point of view, this allows post-experimental choice of the optimal detection potential. Different detection potentials can even be chosen for each chromatographic peak. Having the voltammetric data as well as the chromatographic data provides ready identification of chromatographically unresolved compounds and the ability to resolve such co-eluting compounds voltammetrically. The voltammetric data also provide a second method of peak identification for greater certainty in peak assignments. Voltammetric detection limits of less than 10 pmol of material injected on the column were achieved with this detection method. From the electrochemical perspective, voltammetric/amperometric detection provides a technique for obtaining hydrodynamic voltammograms with small amounts or small volumes of sample. Voltammograms can also be obtained for the individual components of complex mixtures without the need for isolation steps.     

高效液相色谱手性固定相法拆分阿折地平对映体

建立了阿折地平对映体的高效液相色谱拆分方法。采用Chiralpak AD-H (250 mm×4.6 mm, 5.0 μm, Daicel公司)手性色谱柱在正相条件下直接拆分阿折地平对映体,考察了固定相种类、流动相组成及柱温等对阿折地平对映体分离的影响。确定了最佳的拆分条件: 流动相为正己烷-异丙醇(90:10, v/v),流速为0.8 mL/min,检测波长为254 nm;柱温为20 ℃;在此条件下阿折地平对映体的分离度为3.3。该法简单快速,重现性好。     

Specific rotation measurements from peak height data, with a Gaussian peak model

A method is described whereby a sensitive laser-based polarimeter can be used to make very accurate and precise specific rotation measurements on microgram quantities of optically active materials. Flow-injection or liquid chromatography systems provide reproducible introduction of the sample into the polarimetric system. If a Gaussian distribution of the analyte concentration is assumed, the peak height can be used in the determination of specific rotation. This method provides a direct calibration with an absolute standard which yields more accurate and precise results than those obtainable by using peak area.     

Use of evaporative light scattering detector in the detection and quantification of enantiomeric mixtures by HPLC

Routinely used in our laboratories at analytical scale, an evaporative light scattering detector (ELSD) has proved to be versatile in the detection of enantiomeric resolution using chiral stationary phases by HPLC. Though this kind of detector has been widely used in various domains, its application in enantiomeric resolution has not been discussed in the literature and is found to have very specific features especially in the quantitative perspective. In contrast with the UV detection, the peak area from ELSD for both enantiomers of a racemic mixture may not be the same. This complicates the assessment of the enantiomeric purity of unknown samples. This current work deals with some practical aspects in the detection of enantiomers and in accurate quantitative determination of enantiomeric purity by ELSD. Effects of analyte nature (more precisely molecular weight and volatility), peak shape and peak shape difference between enantiomers on the quantitative integration by ELSD are discussed in connection with the UV-detection results. The calibration for quantitative enantiomeric analysis and its effectiveness are demonstrated.     

True or apparent reversal of elution order during chiral high-performance liquid chromatography monitored by a polarimetric detector under different mobile phase conditions

The use of all chromatographic modes (i.e. reversed-phase, normal-phase and polar organic modes) is becoming more and more systematic in chiral HPLC method development. When this method development is monitored by polarimetric detection, which is a widespread technique, one should not forget that the observed positive or negative rotation angle for a given absolute configuration depends on the solvent. As a cautionary illustration, a new example is reported for a racemate which is separated with the same elution order in ethanol or acetonitrile mobile phase on a CHIRALCEL OD-R column whereas the signs of polarimetric detection are reversed.     

Ion-exclusion chromatography of aliphatic car☐ylic acids on a cation-exchange resin by elution with polyvinyl alcohol

Ion-exclusion chromatography of aliphatic car☐ylic acids of different acidity (pK_a) and hydrophobicity was investigated on a polystyrene-divinylbenzene (PS-DVB) based strongly acidic cation-exchange resin in the H⁺ form and conductivity detection by elution with polyvinyl alcohol (PVA). When water was used as an eluent, the resolution of the car☐ylic acids was very low and the peak accompanied a fronting depending on their hydrophobicities. Therefore, to improve the peak shape and the peak resolution, aqueous eluents containing PVAs (degrees of polymerization, n=500, 1500and2000) with many OH groups were tested for the ion-exclusion chromatographic separation of the car☐ylic acids. When aqueous eluents containing PVA were used, the fronting was decreased dramatically by the effect of increased hydrophilicity of the PS-DVB cation-exchange resin surface due to adsorption of OH group in PVA. The high resolution ion-exclusion chromatographic separation without the fronting and highly sensitive conductimetric detection of the car☐ylic acids was accomplished successfully by elution with a 0.2% PVA (n=1500)-10% methanol-water.     

直链淀粉手性固定相法分离利阿唑对映体

建立了利阿唑对映体的高效液相色谱拆分方法。采用Chiralpak AD-H手性柱在正相条件下直接拆分利阿唑对映体,考察了流动相中有机极性调节剂的种类和浓度、酸碱的种类和含量、柱温及流速等对利阿唑对映体分离的影响。确定了最佳的拆分条件:流动相为正己烷-乙醇(含0.3%二乙胺和0.1%冰醋酸)(体积比为80∶20),流速0.6 mL/min;检测波长254 nm;柱温20 ℃。在此条件下利阿唑对映体的分离度为3.4。该法简单快速,重现性好。     

Application of power functions to chromatographic data for the enhancement of signal to noise ratios and separation resolution

Chromatographic detection responses are recorded digitally. A peak is represented ideally by a Guassian distribution. Raising a Guassian distribution to the power ‘n’ increases the height of the peak to that power, but decreases the standard deviation by √n. Hence there is an increasing disparity in detection responses as the signal moves from low level noise, with a corresponding decrease in peak width. This increases the S/N ratio and increases peak to peak resolution. The ramifications of these factors are that poor resolution in complex chromatographic data can be improved, and low signal responses embedded at near noise levels can be enhanced. The application of this data treatment process is potentially very useful in 2D-HPLC where sample dilution occurs between dimension, reducing signal response, and in the application of post-reaction detection methods, where band broadening is increased by virtue of reaction coils. In this work power functions applied to chromatographic data are discussed in the context of (a) complex separation problems, (b) 2D-HPLC separations, and (c) post-column reaction detectors.     

Validated Chiral LC Method for the Enantiomeric Separation of β-Amino-β-(3-Methoxyphenyl) Propionic Acid

A chiral liquid chromatographic method for enantiomeric resolution of β-amino-β-(3-methoxyphenyl) propionic acid was developed and validated. The “hybrid” π-electron donor–acceptor based stationary phase (R,R) Whelk-01 was found to be enantiomerically selective for (R) and (S) enantiomers of β-amino-β-(3-methoxyphenyl) propionic acid with a resolution greater than 2.0. The effects of isopropyl alcohol and ethanol on enantioselectivity and resolution of enantiomers were evaluated. Calibration curves were linear over the range of 0.10–1.00, with a regression coefficient (r) of 0.999. The limit of detection (LOD) and limit of quantification (LOQ) were 300 and 1,000 ng mL⁻¹ respectively for 10 μL injection volume. The percentage RSD of the peak area of six replicate injections of (S) enantiomer at LOQ concentration was 2.8. The percentage recovery of (S) enantiomer from (R) enantiomer samples ranged from 92 to 102. The test solution was observed to be stable up to 24 h after the preparation. The developed normal phase chiral LC method can be used for the enantiomeric purity evaluation of R-β-amino-β-(3-methoxyphenyl) propionic acid.     

HPLC enantioseparation of alkaloid malacitanine using fluorimetric/polarimetric detection

This work reports two methods developed for the separation and determination of the enantiomers of the new alkaloid malacitanine (MLC) and the determination of the enantiomeric purity in mixtures. First, the isomers were separated using a Chirex 3020 (250 mm × 4.6 mm, 5 μm) chiral column with a mobile phase of cyclohexane–1,2‐dichloroethane–ethanol–trifluoroacetic acid (64:30:6:0.6, v/v/v/v) at a flow rate of 1 mL/min and fluorimetric detection. Obtained retention times were 12.4 and 15.9 min (+ and ?) with a resolution Rs of 1.13. Relative standard deviations (RSDs) were 2.5 and 2.4% at the 0.5‐μg level (four determinations). Second, a nonenantioselective procedure for the determination of enantiomeric purity of MLC using a Lichrospher ® Si‐60 (250 mm × 5 mm, 5 μm) normal phase with a mobile phase of 100% ethanol at a flow rate of 0.9 mL/min coupled to two detectors in series, fluorimetric and polarimetric. RSD of 3.3% was obtained. Calculated enantiomeric purity by chiral chromatography gave 48.6% (?)‐MLC in the near racemic product. Using polarimetric signal of the nonseparated enantiomers and comparing the slopes of the calibration curves (enantiomers) from the racemic product gave 47.8% (?)‐MLC content. A study of accuracy of (?)‐MLC gave recoveries from 98.3 to 100.7%.     

Quantification of very low enantiomeric impurity of efaroxan using a dual cyclodextrin system by capillary electrophoresis

A rapid method for the enantiomeric purity determination of efaroxan has been developed by capillary electrophoresis (CE) using a dual cyclodextrin (CD) system. The influence of the nature and the concentration of CDs on separation parameters has been studied. High resolution (R_s = 7) and peak efficiency (104 000-121 000 theoretical plates) values were obtained for efaroxan enantiomers by adding two cyclodextrins, one neutral (7.5 mM DM-β-CD) and the other negatively charged (3 mM CM-β-CD) to the running buffer composed of 100 mM phosphoric acid-triethanolamine (pH 3). These resolution and peak efficiencies values allowed the quantitation of the (S)-enantiomer of efaroxan at very low enantiomeric excess even if the minor component migrates after the major one. This method was fully validated for the enantiomeric impurity determination of the (S)-form of efaroxan at the 0.05% level. Calibration curve, expressed by the peak areas ratio versus the enantiomeric purity was linear over the 0.05-1% enantiomeric impurity range (r² = 0.9996). Limits of detection (LOD) and quantification (LOQ), expressed in term of (S)-enantiomer impurity were 0.02% and 0.05%, respectively. The accuracy of the method at 0.12%, 0.50% and 0.80% enantiomeric impurity levels for the (S)-form were determined. Recoveries were in 94-102% range for each quality control sample and were determined with good precision (intra-day R.S.D. = 3.54%, inter-day R.S.D. = 5.33%).     

Direct enantiomeric separation of saterinone by high-performance liquid chromatography

A new method is described for the direct liquid chromatographic separation and determination of the enantiomers of saterinone, 1-[(4-cyano-1,2-dihydro-6-methyl-2-oxopyridinyl-5)-phenoxy]-3-[4-( 2- methoxyphenyl)piperazinyl-1]-propan-2-ol, using a Chiralcel OD column with methanol as eluent and a temperature set to 10 degrees C. Several lots of synthesized enantiomers were analysed for their enantiomeric purity of more than 99%, because of the excellent resolution (Rs = 2.2). Furthermore the enantiomeric ratio was determined in plasma samples after oral and intravenous administration of racemic saterinone.     

Direct liquid chromatographic resolution of racemic aminoglutethimide and its acetylated metabolite using a chiral alpha 1-acid glycoprotein column

A 10-cm long alpha 1-acid glycoprotein column is used for the enantiomeric resolution of the clinically used racemic aminoglutethimide (+/- AG) and its acetylated metabolite (+/- AAG). A direct liquid chromatographic resolution of racemic aminoglutethimide and its acetylated metabolite is accomplished without any derivatizations. Maximum resolutions of 1.37 and 0.73 are obtained for the enantiomers of aminoglutethimide and its acetylated metabolite, respectively. The effect of the 2-propanol content in mobile phase on retention and enantioselectivity of aminoglutethimide and its acetylated metabolite is demonstrated. The variation of the separation factors (alpha) with pH in enantiomeric separation of aminoglutethimide is also shown.     

On-line microdialysis coupled with microbore liquid chromatography with ultraviolet detection for continuous monitoring of free cefsulodin in rat blood

A microdialysis method followed by a microbore liquid chromatographic ultraviolet detection procedure has been performed for the assay of unbound cefsulodin in rat blood. A microdialysis probe was inserted into the jugular vein for blood sampling. This method involves an on-line design for submitting dialysate into the liquid chromatographic system. The chromatographic conditions consisted of a mobile phase of methanol-100 mM monosodium phosphoric acid (10:90, v/v, pH 5.0) pumped through a microbore reversed-phase column at a flow-rate of 0.05 ml/min. Detection wavelength was set at 265 nm. Microdialysis probes, being laboratory-made, were screened for acceptable in vivo recovery while chromatographic resolution and detection were validated for response linearity as well as intra- and inter-day variabilities. The method was then applied to pharmacokinetics profiling of cefsulodin in the blood following intravenous administration of cefsulodin (20 mg/kg) in rats. Pharmacokinetics were calculated from the corrected data for dialysate concentrations of cefsulodin versus time. Based on pharmacokinetic calculation, cefsulodin best fitted to a two-exponential disposition. This study provided specific pharmacokinetic information for protein-unbound cefsulodin and demonstrated the applicability of this continuous sampling method for pharmacokinetic study.