Recent Advances in Analytical Techniques: Volume 5

Superior Aspects of Liquid Chromatography-Based Mass Spectrometers in Chiral Analysis

Aysegul Dogan¹, Cemil C. Eylem¹, Nursabah E. Başcı¹, *

¹ Faculty of Pharmacy, Department of Analytical Chemistry, Hacettepe University, 06100, Sıhhiye, Ankara, Turkey

Abstract

Chirality has many important roles in life activities because enzymes, amino acids, nucleic acids, carbohydrates, fats, metabolic intermediates, and many other types of biomolecules are chiral. Due to the different properties of enantiomers, chirality is important in biological systems, and it is also critically important in many other fields, such as the pharmaceutical industry, chemical industry, petrochemical industry, food industry, and agrochemicals, particularly, medicine. Roughly 56% of the pharmaceuticals currently in use are chiral, and 88% of these are administered in racemic proportions, while single-enantiomer formulations of some marketed drugs have shown the higher potency of one stereoisomer compared to the other. Although they have the same chemical structures, most of the enantiomers present in racemic drugs have different pharmacokinetic, pharmacodynamic, biological, and toxic effects. The amounts of chiral molecules in different matrices are far below the levels required for the analysis of pharmaceutical preparations. Therefore, high resolution and sensitivity are needed to analyze chiral molecules. Mass spectrometers, which generally offer higher levels of sensitivity than conventional detection systems and accordingly allow the analysis of lower levels of analytes, have made large contributions to separation and detection science. Developments in new types of columns, different analysis modes, different matrices, and pharmaceutics will be explored in this chapter. The parameters will be discussed with the pros and cons together with their applicability to different sample types.

Keywords: Bioanalysis, Biological Material, Chiral Analysis, Chiral Columns, Chirality, Enantiomers, ESI, GC-MS, IM-MS, LC-MS, Mass Spectrometer, MS/MS, Pharmaceutical Preparation, Plasma, QTOF-MS.

* Corresponding author Nursabah E. Başcı: Faculty of Pharmacy, Department of Analytical Chemistry, Hacettepe University, 06100, Sıhhiye, Ankara, Turkey .Tel: +90 (312) 305 10 88; E-mail: [email protected]

INTRODUCTION

Chirality is found in many areas of our life, from living systems to natural and synthetic organic substances. Chirality plays a dominant role in the interaction of molecules with biologically active substances, and it has created a new era in the

development of the science branches. In other words, it has brought a third dimension to all fields of science.

Isomers are two or more different compounds that can be represented by the same molecular formula. These are compounds possessing the same molecular formulas while having different atomic arrangements. For this reason, the chemical structures of isomeric compounds also differ. This phenomenon can be examined within the two categories of structural isomers and stereoisomers. The latter are molecules possessing the same structure and differing only by their atomic arrangements. The covalent bonds and functional groups of a biomolecule are central to the function of that molecule. Stereochemistry is also known as the three-dimensional design of molecules (the prefix stereo- means three-dimensionality), and since the enantiomers of chiral molecules are optically active, they refract polarized light to the right and left. In particular, carbon atoms exist in the form of stereoisomers. Isomers can be divided into conformational and configurational isomers. Enantiomer is the name given to one of two stereoisomers, which is a mirror image of the other [1].

The word chirality has its origins in the Greek word kheir, which refers to the use of the right or left hand. Basically, chirality occurs when all the elements bonded to any element (mostly carbon atoms) are different from each other. A symmetrical center or axis does not exist and these molecules can occur in more than one form. In the most general form, chiral molecules have mirror images that do not coincide [2-4].

Molecules that cannot be superposed and are mirror images of each other, as stated above, are known as enantiomers. Since the enantiomers of chiral molecules are optically active, they refract polarized light to both the left and right. Therefore, there are two types of enantiomers, S-enantiomers and R-enantiomers, where the S and R come from the Latin words sinister and rectus, respectively, which mean left and right. Mixtures containing equal amounts of these enantiomers are called racemic [5, 6].

The development of chirality first started with the disintegration of the crystal molecule by Hauy in 1809. It became, even more, focal in 1848, when Louis Pasteur identified two crystals whose mirror images were asymmetric during the examination of tartaric acid crystals. Dr. J.H. van’t Hoff discussed the specific arrangement of the four groups around the central carbon atom. Over time, the importance of these studies began to increase, and van’t Hoff became the first scientist to receive a Nobel Prize in chemistry in 1901. In 1883, the concept of chirality had been fully expressed by Lord Kelvin with the following definition: If it does not coincide with the mirror image of a geometric shape or a group of points itself, it is called chiral [7].

Chiral compounds have been widely used in industrial applications due to their various advantages. The reason why chirality is important for biological activity is that symmetry at the molecular level is dominant in biological processes [8]. In this sense, stereochemistry, the production of pharmaceutical products, and chiral properties are very prominent in determining the pharmacological effects of drugs. In particular, there is now significant interest in chiral separation for isolating and studying enantiomers. The chirality of molecules is important in the pharmaceutical field as well as in agriculture, food, electronics, and other applications. In addition to amino acid, enzyme, and hormone structures, chirality is also important in the plant, animal, and human life, and these molecules can be detected in living things by various methods, especially chromatographic methods.

Chiral drugs can display diverse characteristics in terms of their bioavailability, metabolization, distribution, and elimination, and they also possess qualitatively and quantitatively variable pharmacological and toxicological properties [9-11]. This attracts attention in the pharmaceutical market because of its superiority. Efforts to develop novel approaches for enantioselectively producing new chiral compounds are widely supported.

In the drug market, 88% of the drugs sold are mixtures consisting of racemates. Although they have identical chemical structures, the enantiomers present within racemic drugs generally possess varying characteristics in terms of their pharmacokinetics, pharmacodynamics, and biological and toxic effects. In its relevant guidelines, the US Food and Drug Administration (FDA) emphasizes that the physical effects of the different enantiomers present within racemic drugs need to be evaluated individually and that the design of novel chiral compounds as single enantiomers should be pursued [12]. It is possible that the enantiomers constituting drugs’ active ingredients are different and more effective than the isomers. For example, in a finding now known in the medical literature as the thalidomide disaster, thalidomide’s S-enantiomer exerts teratogenic effects, while the R-enantiomer has sedative properties [13]. Verapamil’s R-enantiomer has applications as a multidrug resistance modulator in chemotherapeutics for the treatment of cancer, and the S-enantiomer has applications as a calcium channel blocker. At the same time, the R-enantiomer has been demonstrated to exert cardiotoxic effects [7]. Increases in cases like these have prompted researchers to consider drug molecules as chiral molecules and to focus on the production of single-enantiomer molecules whenever possible.

Gas chromatography, high-performance liquid chromatography (HPLC), supercritical liquid chromatography, and capillary electrophoresis are widely utilized for separating chiral pharmaceuticals throughout the stages of drug discovery. These electrophoretic and chromatographic techniques are highly valuable in the routine determination of enantiomeric purity in molecules that will be produced for pharmaceutical purposes. Among these various approaches, liquid chromatography is one of the most commonly used due to the considerable numbers of different columns on the market, the ranges of selectivity that are available, and the simplicity of scalability up to the level of preparation of analytical results. Especially for enantiomer separations of pharmaceutical molecules, more than 80% of multiple-system solvents in the polar organic phase, normal phase, or reverse-phase show successful results with polysaccharide-derivative fixed stationary phases (cellulose, amylose).

Steadily growing numbers of novel molecules are being produced today as drug candidates, and when enantiomeric purity of such molecules is routinely determined before they are developed any further, the pharmaceutical industry is accordingly encouraged to pursue the concurrent development of rapid chiral analysis methods that follow straightforward protocols. The speed of such analytical procedures is of crucial importance. Therefore, screening strategies like supercritical fluid chromatography (SFC) and HPLC are applied, involving a process of selecting limited numbers of chiral selectors capable of powerful chiral recognition.

A significant proportion of commercial and investigational pharmaceutical molecules are enantiomeric in composition, and a considerably large proportion of those show important enantioselective variation both pharmacokinetically and pharmacodynamically. Recognition of the importance of drug chirality has been growing accordingly, and the application of such molecules as enantiomers or racemates is now frequently explored in the drug literature. As evidence of the problems related to stereoselectivity in drugs has also increased, enantioselective analytical methods together with chromatographic tools have become a central point of the research being performed by many scientists. The separation of chiral compounds is of paramount importance because most bioorganic molecules possess chirality.

In 2001, William S. Knowles, K. Barry Sharpless (from the USA), and Ryoji Noyori (from Japan) were awarded the Nobel Prize in chemistry in recognition of their work on catalytic asymmetric synthesis. This work offered unique insights into how chiral molecules can be used to accelerate and control important

chemical reactions. It was stated that the results of their studies would lead to discoveries regarding antibiotics, new heart drugs, and better control of chemical reactions that will open the door to the treatment of Parkinson’s disease.

Combined instrumental analysis techniques via high-pressure liquid chromatography and gas chromatography are very popular among researchers who are pursuing the separation of enantiomers. These techniques may be used in combination with modern MS detectors that offer high levels of sensitivity (GC-MS/MS, GC-MS, and LC-QTOF-MS). While gas chromatography often finds usage in efforts to separate volatile components, it is also suitable for qualitative and quantitative analysis [14]. The Chiraldex® GPN capillary GC column is also used for the stationary phase. Capillary Chiraldex® GPN GC columns provide phases consisting of α-, β-, or Ɣ-cyclodextrin derivatives for the separation of enantiomers. These columns are capable of separating various non-derivatized enantiomers that are difficult to resolve by HPLC. These columns selectively and effectively separate most of the enantiomeric molecules in chiral synthesis, biochemical and pharmaceutical molecules, environmental pollutants, sweeteners, and many other matrixes (Fig. 1).

Fig. (1))

Flow chart for chiral analysis.

In liquid chromatographic enantiomeric separations, the separations are performed using chiral stationary phases. The Chirobiotic® V2 column is based on the binding of vancomycin with 18 chiral centers to high-purity silica gel surrounding three wells. These chiral stationary phases will undergo multiple molecular interactions with analytes that are polar, ionizable, and neutral. In other words, this system is quite versatile and a single Chirobiotic® column may be used with success in various mobile phases, allowing operation in normal or reverse phase. While this is valuable, an even more important feature of these Chirobiotic® columns is that ionic interactions occur, which allow for their application in both polar ionic and reverse-phase modes to conduct precise liquid chromatography-based mass spectrometry work. Six different molecular interactions take place in these columns, H-bond, ionic, P-P, hydrophobic, dipole, and steric. The use of these columns also offers multiple inclusion sites, which affect the selectivity according to the chosen analytes’ molecular shapes. Enantiomer separation can be optimized by making changes to the mobile phase, affecting both the type and the relative strength of different interactions. The most important feature of these columns is that they may be operated in normal, reverse and polar organic phases. Internal diameters of chiral columns range from micrometers to 10-20 cm. The reduction of the column diameters allows for lower solvent consumption, miniaturization with low sample volume, and less harmful waste for the environment while providing acceptable green chromatographic separations. The stationary and mobile phases used in liquid chromatographic studies are more interesting in terms of green analytical chemistry compared to gas chromatographic methods, as they have aspects that need to be greened more in terms of analysis parameters and the solvent and methods used in sample preparation, and they are among the methods frequently preferred by analysts.

MASS SPECTROMETRY IN CHIRAL ANALYSIS

Many biomolecules, such as enzymes, amino acids, fats, nucleic acids, carbohydrates, and metabolic intermediates, which are common in living organisms and have important roles in many vital activities, have chiral properties. In addition to biological systems, chiral analysis is important in many other fields, including chemistry, petrochemical, and agrochemical industries, and especially in the field of pharmaceuticals, as the optical isomers of chiral drugs cause significant differences in pharmacological and toxicological activities [15]. Especially in applications in the field of medicine, approximately 80% of medicinal chemicals and over 50% of drugs used today possess chirality, while about 90% of the drugs currently being marketed are racemic mixtures [7, 16]. Therefore, developing and implementing efficient chiral analysis methods is critically important, especially in terms of life and environmental science. Enantiomer-based analysis of chiral compounds remains a challenging topic in many areas, such as environmental, pharmaceutical, and biomedical applications. Analysis of chiral compounds for categorization as (+) and (-) enantiomers is very challenging because enantiomers have the same physical and chemical properties [17].

The ultraviolet (UV) detector, traditionally used in liquid chromatography (LC), was the most widely used technique for chiral analysis. In addition, detectors with different operating principles, such as fluorescence, conductivity, and refractive index have been used in the chiral analysis. Nowadays, mass spectrometry (MS) is often preferred for chiral analysis as it gives better analysis speed, cost-effectiveness, and selective, sensitive, and accurate results [18, 19].

MS is a powerful approach to analysis, and it offers valuable benefits, including high sensitivity, increased speed, and good molecular selectivity for the analysis of molecules that have different chemical groups [20, 21]. As a result of the development of modern instruments, MS began taking an increasingly central position in chiral analyses with its capability of analyzing small molecules of less than 2 ppm (0.0002%) while being highly selective and having a very low mass error [22]. Mass spectrometers are used for the separation and detection of analyte ions through their mass-to-charge ratios, while extensive databases, such as PubChem [23], METLIN [24], and HMDB [25], are used to identify molecules in non-targeted analyses, and analyte standards are often used in targeted analyses. In the beginning, MS was considered a chiral-blind technique since chiral molecules are defined by the rotation of plane-polarized light and enantiomers will possess the same masses and yield the same mass spectra [26, 27]. This limitation was due to the inability of MS to distinguish a difference in mass between the two enantiomers of a chiral molecule. By increasing molecular masses, structural complexities also increase; consequently, the probability of detecting more than one isomeric species with a common molecular mass increases. It accordingly becomes very difficult to separate and identify molecules with similar structures when the potential isomers of an analyte increase in quantity [26]. Thus, MS is often used combined with chromatographic techniques, such as capillary electrophoresis [28], supercritical chromatography [29], gas chromatography (GC) [30], and LC [31] to overcome the challenge of separating [32] and analyzing chiral molecules.

In recent years, ion mobility and qTOF mass spectrometry, detecting mass at high resolution, has become a popular analytical approach due to its ability to distinguish isomeric species in complex biological samples [33]. However, the applications are still limited and there are few relevant studies in the literature [34, 35]. On the other hand, the ability to apply various fragmentation techniques by tandem mass spectrometry (MS/MS) is often utilized, as it is quite powerful in terms of its ability to provide an approach to distinguish the variation in fragment ion masses or bond dissociation energies of structural isomers [36, 37]. The analysis of chiral isomers with more similar structures by MS/MS poses a great analytical challenge, however, because the molecules show similar fragmentation [26]. Therefore, MS/MS must be combined with a chromatography technique in chiral separation. Of these possible techniques, LC-MS/MS and GC-MS/MS are the most commonly preferred methods for the analysis of chiral substances in applications from environmental analysis to biological sample analysis due to their high selectivity, sensitivity, and low detection limits [38, 39]. In cases where chiral analysis by GC-MS or LC-MS is not possible, supercritical fluidic chromatography-mass spectrometry (SFC-MS) has been proposed as an alternative analytical technique [40], and it was shown that SFC-MS was superior compared to the normal phase LC-MS technique [41].

In this section, liquid chromatography-based mass spectrometry (LC-MS) and gas chromatography-based mass spectrometry (GC-MS) has been discussed, which are predominant techniques in clinical, pharmaceutical, and environmental chiral analysis. In the literature, it is seen that a wide range of chiral molecules from carboxylic acids to amino acids and from lipids to drug active ingredients were analyzed from complex samples, such as plasma [42], urine [43], tissues [44], serum [45], cell cultures [46], hair [47], and saliva [48], and these studies are presented in Table 1.

Table 1 Chiral analysis as performed by GC-MS and/or LC-MS based on biological materials.