Michael E. Swartz

UPLC™: An Introduction and Review

Abstract Ultra performance liquid chromatography™ (UPLC) takes advantage of technological strides made in particle chemistry performance, system optimization, detector design, and data processing and control. Using sub‐2 µm particles and mobile phases at high linear velocities, and instrumentation that operates at higher pressures than those used in HPLC, dramatic increases in resolution, sensitivity, and speed of analysis can be obtained. This new category of analytical separation science retains the practicality and principles of HPLC while creating a step‐function improvement in chromatographic performance. This review introduces the theory of UPLC, and summarizes some of the most recent work in the field.

Novel chiral surfactant for the separation of enantiomers by micellar electrokinetic capillary chromatography.

A novel chiral surfactant was prepared as both enantiomeric forms, (R)- and (S)-N-dodecoxycarbonylvaline, and employed for the separation of enantiomeric mixtures by micellar electrokinetic capillary chromatography (MECC). The enantioselectivities (alpha) obtained for twelve typical pharmaceutical amines using the (S)-surfactant were compared to those obtained with (S)-N-dodecanoylvaline, a chiral surfactant described in the literature. Higher enantioselectivities were seen for ten of the twelve compounds using (S)-N-dodecoxycarbonylvaline. Furthermore, (S)-N-dodecoxycarbonylvaline had significantly less background absorbance in the low UV. It is shown that exact enantiomer migration order reversal can be obtained by individually employing both enantiomeric forms of the surfactant. For ionizable compounds like the amines examined here, enantioselectivity can be optimized by changing the pH of the MECC buffer. Partitioning is optimized through surfactant concentration, organic additives and pH. The ability to achieve fast chiral separations is shown. A separation of ephedrine enantiomers in urine is shown, with the only sample preparation being filtration.

On-line sample preconcentration on a packed-inlet capillary for improving the sensitivity of capillary electrophoretic analysis of pharmaceuticals

Abstract An on-line preconcentration method is reported that improves the sensitivity of capillary electrophoretic analyses by at least two orders of magnitude. The method uses a concentrator capillary with a 75 μm I.D. and a 1-mm length packed bed at the inlet end that can concentrate samples using the principles of liquid chromatography. Using low-molecular-mass pharmaceutical standards as examples, the parameters used in developing a preconcentration sensitivity enhancement method were optimized. The optimized method was then used to evaluate the quantitative aspects of capillaries of this type, including run to run and capillary to capillary reproducibility, linearity, and efficiency and resolution. In addition, the analysis of a urine sample spiked with doxepin at the 500 ppb level is reported.

Method Development and Selectivity Control for Small Molecule Pharmaceutical Separations by Capillary Electrophoresis

Abstract This manuscript outlines an approach for capillary electrophoresis (CE) method development. It is applied to small molecule pharmaceutical separations using analgesics as examples. the effect of various parameters on selectivity are evaluated, and some recommendations are made regarding successful quantitation. Such parameters as linearity, sensitivity, and reproducibility are also examined. This knowledge is extended to other application areas, and examples of CE separations for such compounds as penicillins, water soluble vitamins, and enantiomeric compounds are presented. It is shown that the selectivity, sensitivity, and reproducibility of CE are adequate for routine use in the pharmaceutical laboratory.

Validation of enantiomeric separations by micellar electrokinetic capillary chromatography using synthetic chiral surfactants

Abstract The first report on the feasibility of method validation for the separation of enantiomeric mixtures by chiral micelle electrokinetic capillary chromatography is described. Method validation data elements were investigated according to U.S. Pharmacopeia protocol and are reported for the separation of ephedrine enantiomers using the synthetic chiral surfactants ( S )- and ( R )-N-dodecoxycarbonylvaline as buffer additives. Excellent linearity from 1–150% of the target concentration was obtained. A 1.0% limit of quantitation for an enantomeric impurity with acceptable precision is shown. Method robustness and ruggedness is also presented. Migration order reversal was used for the determination of a trace enantiomeric impurity, and to identify the enaniomeric compound in a multiple active ingredient formulation. The capability to reverse migration order is shown to be a valuable tool to satisfy the U.S. Pharmacopeia specificity requirement as well as improving detection and quantitation limits.

Analytical Method Development and Validation for the Academic Researcher

INTRODUCTION During the course of our scientific careers, and after reading numerous original research papers and reviews submitted for publication to various journals or books, it has become apparent to us that many, if not most, academic researchers have little use for method validation studies or demonstrations. Now, that may sound a bit harsh or a strong way to start this mini-review, but this situation has been our collective impression after carefully reading and commenting on hundreds of submitted journal manuscripts over perhaps the past 25 years.

Separation of piperidine-2,6-dione drug enantiomers by micellar electrokinetic capillary chromatography using synthetic chiral surfactants

Abstract The enantioselective separation of five racemic piperidine-2,6-dione compounds was accomplished using chiral micelle electrokinetic capillary chromatography. This class of drugs includes glutethimide, aminoglutethimide, cyclohexylaminoglutethimide, pyridoglutethimide, and phenglutarimide. The resolution of all five compounds with simultaneous enantioselective separation of four of the five was obtained in a single run, using the synthetic chiral surfactant (S)-N-dodecoxycarbonylvaline. An enantioselective separation of the fifth compound was obtained by a second synthetic chiral surfactant, (R)-dodecoxycarbonylproline. It was observed that surfactant type and concentration, pH, and sample matrix all effect enantiomeric resolution. The migration order of cyclohexylaminoglutethimide enantiomers was confirmed by injecting a sample comprised mostly of the (−)-enantiomer. In addition, the separation of a sixth related sample (a thalidomide mixture) is shown to be achiral and not enantiomeric, as verified by the lack of enantiomer migration order reversal.

Capillary electrophoretic determination of inorganic ions in a prenatal vitamin formulation

Abstract Methods for the quantitative analysis of three cations (calcium, iron and zinc), and the qualitative analysis of several anionic species (chloride, sulfate, nitrate, citrate, fumerate, phosphate, carbonate and acetate) from a prenatal vitamin formulation by two different capillary ion electrophoresis methods are reported. The cation method was evaluated for detection and quantitation limits, precision, accuracy and linearity. For standard solutions, detection limits into the ng/ml range, and reproducibility that averaged less than 1% for migration time and 2% for area response were generated. Calibration plots exhibited linearity in excess of three orders of magnitude. In addition, excellent agreement between capillary ion electrophoresis and flame photometry quantitative results for the cation analyses were obtained.

Use of mathematically enhanced spectral analysis and spectral contrast techniques for the liquid chromatographic and capillary electrophoretic detection and identification of pharmaceutical compounds

The use of mathematically enhanced ultraviolet/visible (UV/VIS) absorbance spectral analysis and spectral contrast software techniques in high performance liquid chromatography (HPLC) and micellar electrokinetic capillary electrophoresis (MECC) as an aid for the determination of peak homogeneity, identification, and tracking during method development was investigated. Various structurally similar pharmaceutical compounds, and compounds present as either cis/trans isomers, diastereomers, or enantiomers were used as test compounds to probe the limits of this technique. Two tricyclic antidepressants, nortriptyline and imipramine, were employed to study the effects of HPLC mobile phase composition and pH on the ability to identify and track peaks during method development. It was found that method changes altered the spectral matches used for identification, but not enough to cause incorrect peak identification. It was also shown using HPLC that the cis/trans isomers of doxepin and the diastereomers ephedrine and pseudoephedrine could be distinguished. The mathematically enhanced spectral analysis and spectral contrast software techniques were also employed with MECC. Peaks tracking during method development as pH and the concentration of surfactant changes is shown for a separation of various penicillin type antibiotics. It was shown that during chiral MECC (CMECC) analyses ephedrine/pseudoephedrine diastereomers as well as ephedrine enantiomers could be distinguished. The determination of enantiomers is possible in CMECC since enantiomers are eluted as diastereomeric complexes, as opposed to HPLC where they are eluted in their native state.

6 Contemporary liquid chromatographic systems for method development

Abstract There are several basic components to a liquid chromatographic (LC) system used for method development, and modern technology, from column chemistry to advanced detection and automation, is quickly changing the landscape of method development. The way in which solvents and samples are managed, and the types of columns and detectors used can have a significant impact on both the throughput (time it takes to develop a method) and the quality and/or robustness of the method. This chapter will begin with a brief description of a basic high performance liquid chromatography (HPLC) method development system, and then discuss in some detail modern ultra performance liquid chromatography (UPLC™) instrumentation components and how they are used to develop methods for the types of complex samples encountered in todays pharmaceutical laboratory. Recent advances in UPLC technology, capitalizing on sub-2 μm particle column packings, automated approaches, and migrating from HPLC to UPLC will also be addressed.