Analytical Techniques in Biosciences: From Basics to Applications

By Chukwuebuka Egbuna

Analytical Techniques in Biosciences: From Basics to Applications presents comprehensive and up-to-date information on the various analytical techniques obtainable in bioscience research laboratories across the world. This book contains chapters that discuss the basic bioanalytical protocols and sample preparation guidelines. Commonly encountered analytical techniques, their working principles, and applications were presented. Techniques, considered in this book, include centrifugation techniques, electrophoretic techniques, chromatography, titrimetry, spectrometry, and hyphenated techniques. Subsequent chapters emphasize molecular weight determination and electroanalytical techniques, biosensors, and enzyme assay protocols. Other chapters detail microbial techniques, statistical methods, computational modeling, and immunology and immunochemistry.

The book draws from experts from key institutions around the globe, who have simplified the chapters in a way that will be useful to early-stage researchers as well as advanced scientists. It is also carefully structured and integrated sequentially to aid flow, consistency, and continuity. This is a must-have reference for graduate students and researchers in the field of biosciences.

• Presents basic analytical protocols and sample-preparation guidelines
• Details the various analytical techniques, including centrifugation, spectrometry, chromatography, and titrimetry
• Describes advanced techniques such as hyphenated techniques, electroanalytical techniques, and the application of biosensors in biomedical research
• Presents biostatistical tools and methods and basic computational models in biosciences

• Language: English
• Publisher: Academic Press
• Release date: Oct 21, 2021
• ISBN: 9780128227992

Book preview

Chapter 1

Bioanalysis: methods, techniques, and applications

Mithun Rudrapal¹, Aniket P. Kothawade², Shahira M. Ezzat³, ⁴ and Chukwuebuka Egbuna⁵, ⁶,    ¹Department of Pharmaceutical Chemistry, Rasiklal M. Dhariwal Institute of Pharmaceutical Education and Research, Pune, India,    ²Sandip Institute of Pharmaceutical Sciences, Sandip Foundation, Nashik, India,    ³Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Cairo, Egypt,    ⁴Faculty of Pharmacy, MSA University, Cairo, Egypt,    ⁵Nutritional Biochemistry and Toxicology Unit, World Bank Africa Centre of Excellence, Centre for Public Health and Toxicological Research (ACE-PUTOR), University of Port Harcourt, Port Harcourt, Nigeria,    ⁶Department of Biochemistry, Faculty of Natural Sciences, Chukwuemeka Odumegwu Ojukwu University, Uli Campus, Uli, Nigeria

Abstract

Bioanalysis describes quantitative estimation of chemicals or drug substances as well as their metabolic products in large variety of bio-samples. It is an integrated technique that has been employed in preclinical stages of drug-discovery process to further support the clinical phases of drug discovery. However, a bioanalytical method must be optimized, characterized, and validated following documented procedures according to United States Pharmacopeia (USP)/International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines to comply with the regulatory guidelines and acceptance criteria. Bioanalytical studies should therefore provide accurate and reproducible estimation of drugs or metabolites in biological samples at great level of sensitivity, selectivity, and specificity. In this chapter, various advanced techniques commonly employed in bioanalysis have been summarized. Different techniques of extraction and an array of advanced hyphenated techniques are employed for the evaluation of bioanalytes in biofluids, with improved analytical specificity and sensitivity. The applications of bioanalysis in biomedical, pharmaceutical, and other allied areas have been systematically reviewed in this chapter.

Keywords

Bioanalytical; hyphenated techniques; method development; validation; pharmacokinetics

Abbreviations

ADME Absorption, distribution, metabolism, and excretion

APCI Atmospheric pressure chemical ionization

BA Bioanalysis

CE-MS Capillary electrophoresis-mass spectrometry

ESI Electrospray ionization

ESI-MS Electrospray ionization-mass spectrometry

FAB Fast atom bombardment

FDA Food and drug administration

GC-IR Gas chromatography-infrared spectroscopy

GC-MS Gas chromatography-mass spectrometry

GLP Good laboratory practices

ICH International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use

LC-MS/MS Liquid chromatography-mass spectrometry/mass spectrometry

LC-MS Liquid chromatography-mass spectrometry

LC-NMR Liquid chromatography-nuclear magnetic resonance spectroscopy

LOD Limit of detection

LOQ Limit of quantification

MALDI Matrix-assisted laser desorption ionization

RSD Relative standard deviation

1.1 Introduction

Bioanalysis (BA) involves the estimation of the concentration of analyte (or drug substances) as well as their metabolic products in biological samples/specimens such as plasma, urine, serum, and blood. It is primarily a technique for the analysis of various forms of analytes (drugs, metabolites, enzymes, or biomarkers) within a biological sample [1]. Hyphenated techniques (combination of chromatographic and spectroscopic/spectrometric methods) are mainly used for analytical determinations (detection and quantification) in biosamples. BA is an integrated technique that has been employed in preclinical stages of drug-discovery process to further support the clinical phases of drug candidates. It also plays a vital role in pharmacokinetics (PK), metabolic fate/drug metabolism, toxicokinetics, pharmacodynamics (PD), bioavailability, and bioequivalence studies [2–4]. BA has many other applications in forensic science, food science, clinical (bio)chemistry, and biomedical or life science [5,6]. Apart from investigation of drugs or metabolites, BA also analyzes macromolecules or biomolecules such as peptides, proteins, and nucleic acids [7]. However, any method of BA must be optimized (after development), characterized, and validated following documented procedures according to USP/ICH guidelines to comply with the acceptance criteria as well as meet the prescribed standards. Bioanalytical studies should, therefore, provide accurate, reliable, and reproducible estimation of drugs or metabolites in biological samples in great level of sensitivity, selectivity, and specificity [8–10].

1.2 Bioanalytical methods

A method in BA could be a group of all techniques that are involved with the collection, storage, analysis, and processing of the biological matrix/sample of an analyte. The procedure for quantifiable estimation of drugs and their metabolic products in BA includes the following steps: sampling, sample preparation, sample analysis (separation, identification, detection, and quantification), calibration, data evaluation, and finally reporting and storage of information [11] (Fig. 1.1).

Figure 1.1 Steps involved in bioanalysis.

A bioanalytical method comprises of the two major steps as described further.

1.2.1 Sample preparation

The preparation of sample involves preliminary cleanup of the biological sample (usually removal of protein from biological fluids) such as biological fluids like urine, serum, and plasma before performing the actual analysis. The extraction methods frequently employed in processing biological sample involve the two steps, viz., extraction and concentration [12,13]. Extraction involves the separation and isolation of analytes (drugs/metabolites) from the biological sample. The concentration of sample is done to enhance the precision of the analytical technique.

1.2.2 Sample measurement

The analyte(s) are quantitatively measured or detected in the biological samples with the aid of instrumental analytical techniques such as high-performance liquid chromatography (HPLC), mass spectrometric techniques, or hyphenated techniques [Liquid chromatography-mass spectrometry (LC-MS), Gas chromatography-mass spectrometry (GC-MS), Liquid chromatography-mass spectrometry/Mass spectrometry (LC-MS/MS), etc.]. The sample could be detected with high specificity and sensitivity [14,15].

1.3 Sample preparation

Biological samples are biological specimens of complex biological matrix. Blood, plasma, serum, urine, feces, gastric contents, saliva, sputum, bile, seminal fluid, CSF, skin, hair, tissue homogenates, or tissue extracts represent the most common biological samples that usually need to be analyzed. The concentration of analyte/drug remains usually very low [milligrams per milliliter (mg/mL), nanogram per milliliter (ng/mL), or less] in biological samples. Additionally, drug is often bound by proteins/tissues (or covalently conjugated) to form a metabolite which is more water soluble, which makes the matrix of sample complex in nature. Hence prior to method development, the sample preparation must be done to remove many interfering substances such as proteins, fibrins, and blood cells that forms the biological matrix [12,16–19]. Fig. 1.2 presents some techniques of sample preparation frequently employed in bioanalytical studies.

Figure 1.2 Techniques of sample preparation. DLLME, Dispersive liquid–liquid microextraction; LLE, liquid–liquid extraction; SPE, solid-phase extraction; PPT, protein precipitation technique; SPME, solid-phase microextraction; SDME, single-drop microextraction.

Sample preparation techniques [liquid–liquid extraction (LLE), solid-phase extraction (SPE), and protein precipitation technique (PPT)] are described briefly as follows. A comparison between LLE, SPE, and PPT is enlisted in Table 1.1.

Table 1.1

1.3.1 Liquid–liquid extraction

LLE is also known as solvent extraction technique, which stands on the principle of differential solubility and partition equilibrium of a solute or an analyte between an organic solvent (nonpolar) and an aqueous solvent (polar). It aims at purifying the analyte by the approach of partitioning the components of sample between two distinguished immiscible solvents [11,20]. This method is user-friendly, quicker, and more reasonable in comparison with other methods. LLE is frequently employed in sample preparation for cleanup of different biological matrices, including blood, serum, urine, etc., that are analyzed by LC-MS or GC-MS.

1.3.2 Solid-phase extraction

This extraction technique utilizes physical and chemical properties of analytes that are diffused in a solvent to isolate them from other interferences. This technique is believed to be an important technique used widely in the preparation of samples for bioanalytical studies. It usually takes place between a solid and a liquid phase. In this method, the preparation of sample involves four steps, viz., conditioning, sample loading, washing, and elution. It is more sophisticated method than LLE technique. In this method, high extraction output is obtained from the bioanalyte [12–14].

1.3.3 Protein precipitation

PPT is another useful method that employs some selective organic solvents having good solubility and protein precipitating properties for samples preparation in LC-MS for BA. Such technique fits dealing with plasma or blood samples with high analyte concentration [15]. In this method, biological samples are mixed with protein precipitating agent followed by dilution with solvent (3–5 times the sample amount). Methyl alcohol and acetonitrile or acidified solutions such as diluted trifluoroacetic acid are solvents of choice due to their desired solubility in addition to precipitation properties. PPT is commonly used for quick cleanup and isolation of drugs/metabolites from blood samples, which include serum, plasma, or whole blood during BA. There are two types of PPT methods, one is salting out, in which ammonium sulfate is used as protein precipitating agent, and the other one is solvent precipitation, where water-miscible solvents like ethanol, methanol, or acetone are used as protein precipitating agents [12,13].

1.3.4 Microextraction techniques

Microextraction is a modern extraction technique where the ratio of extracting solvent to the sample volume is very low and the analytes isolation is usually not exhaustive. Microextraction is a relatively new and efficient technique for sample preparation. The practice of using microextraction techniques in BA has certain advantages such as high throughput sample preparation, increased extraction efficiency, small sample volume, precision, reduced solvent consumption, and low cost. Some widely used techniques are solid-phase microextraction, liquid-phase microextraction, single-drop microextraction, microextraction by packed sorbent, and dispersive liquid–liquid microextraction [16,17].

Prior to actual analytical determination, the preparation of sample is necessary due to the following points [19,21]:

• elimination of undesired components (primarily proteins) that may interfere with the estimation of analyte,

• analyte concentration that depends on the apparatus detection limit,

• extraction of a selected analyte, and

• elimination of unwanted components to protect the chromatographic column and detector.

To make a better choice, one should also consider the recovery of the analyte to assess the effectiveness of a sample preparation technique. This is known as extraction efficiency. The recovery of an analyte from a sample matrix means the difference between the quantity detected of the analyte spiked to and isolated from the sample matrix (preextraction spike) with the amount detected in a postextraction spike. The efficiency of an extraction should be ≤100% [22–24].

1.4 Bioanalytical techniques

Analytical techniques that are utilized in bioanalytical studies include different chromatographic and spectroscopic/spectrometric techniques (Table 1.2). Chromatographic methods are employed as separation technique, while spectroscopic analyses are employed as a technique of detection.

Table 1.2

In recent times, the combined technique of an isolation approach with spectroscopic detection techniques, known as coupled techniques are frequently applied in bioanalytical purposes [1] (Fig. 1.3). Other tools such as capillary electrophoresis (CE) or its hyphenated form, capillary electrophoresis-mass spectrometry (CE-MS), and ligand binding assays (LBA or LBA-LC-MS) are also used in BA.

Figure 1.3 Chromatographic-spectroscopic hyphenated techniques.

1.4.1 Hyphenated techniques

A hyphenated technique involves co-joining of more than one analytical system with the aid of suitable interface. Hyphenated techniques can be called squad analytical techniques too. Several different analytical techniques, chromatographic and spectroscopic techniques, are combined together in a tandem arrangement. The combination of the isolation system with an online spectroscopic tool yields a hyphenated technique [25–27]. In a hyphenated technique, the isolation of the analyte is brought about through a chromatographic column which is then detected by the spectroscopic detector. Hyphenated techniques may be of double or triple hyphenated techniques. Chromatographic methods produce pure or semipure mixtures of the chemical compounds and then the spectroscopy utilizes certain data to identify these compounds [28,29]. Hyphenated techniques range from a mixture of separation and detection (e.g., LC-MS, GC-MS), separation-identification/identification (e.g., LC-MS/MS, GC-MS/MS), and separation/separation-identification/identification (LC/LC-MS/MS) techniques (Fig. 1.4) [30–33].

Figure 1.4 LC/LC-MS/MS. LC, Liquid chromatography; LC-MS, liquid chromatography-mass spectrometry; MS, mass spectrometry.

The distinguished features between LC-MS and GC-MS are presented in Fig. 1.5.

Figure 1.5 Distinguished features between LC-MS and GC-MS. GC-MS, Gas chromatography-mass spectrometry; LC-MS, liquid chromatography-mass spectrometry.

The reasons behind hyphenation are to enhance the responsiveness of the detector, selectivity of separation, specificity of separation, and improve reliability of identification. The advantages of hyphenated techniques are as follows [34,35]:

• speeding up the analysis and less analysis time

• accuracy in measurement

• precise results

• better reproducibility

• analysis of low concentration of analyte(s)

• elucidation of complex structure

• separation and quantification can be achieved

• higher degree of automation

• sophisticated sampling and further analysis

Some specific hyphenated techniques used widely in BA are depicted further.

1.4.1.1 Liquid chromatography-mass spectrometry

The coupling of HPLC with the MS produces a hyphenated technique called LC-MS. The isolated compounds from the LC column are subsequently identified or detected by the mass detector and results of analysis are interpreted with the aid of mass spectroscopy (Fig. 1.6). A typical LC-MS essentially includes an LC system, an interface, and a mass spectrometer [36–38]. The combination of MS to LC possesses improve sensitivity and specificity. The data obtained from LC-MS analysis are crucial for identification and quantification of the analyzed compounds. LC-MS technique is selectively applied for the monitoring of impurity profiling of drugs and metabolites. In LC, the isolation is attained by reversed-phase mode employing either a gradient or an isocratic mobile phase. The ionization techniques utilized in MS component are generally soft ionization methods such as electrospray ionization (ESI) and atmospheric pressure ionization. [39,40]. Mass spectrometers with quadrupole (single or triple), time-of-flight (TOF), ion trap and hybrid are used in LC-MS. Such technique is utilized for analysis of various biological molecules, analysis of vitamins and related metabolites, steroid hormones, therapeutic drug monitoring, and toxicological studies, while the use of tandem MS allows highly sensitive and accurate analysis. Due to the evolution of new low-cost and attested instruments, LC-MS is now playing a crucial role in many aspects of clinical biochemistry being highly competitive to other conventional techniques such as HPLC and immunoassay