Development of bioanalysis: a short history

Abstract

BioanalysisVol. 1, No. 1 EditorialFree AccessDevelopment of bioanalysis: a short historyHoward HillHoward HillSenior Editor, Bioanalysis Director of Pharmaceutical Analytical Services, Huntingdon Life Sciences, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE16 5HS, UK. Search for more papers by this authorEmail the corresponding author at hillh@ukorg.huntingdon.comPublished Online:27 Mar 2009https://doi.org/10.4155/bio.09.3AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInReddit Historically, the initial drivers to measure the presence of drugs in biological fluids were to determine possible overdosing as part of the new science of forensic medicine/toxicology. The need to measure drug levels in biological fluids was further driven by the development of pharmacokinetics as a science in the 1930s.In the early days of drug development, many of the assays for drugs in biological fluids were nonspecific and did not discriminate between the drug and its metabolites; for example, aspirin (circa 1900) and sulfonamides (developed in the 1930s) were quantified by the use of colorometric assays, while antibiotics could be quantified by their ability to inhibit bacterial growth. The 1930s also saw the rise of pharmacokinetics – a driver for more specific assays.The lack of specificity became a real issue as the identification of metabolites gathered speed for drugs in biological fluids. Their development as drugs in their own right made identification a commercial issue, as metabolites could be patented as ‘new’ drug entities. An understanding of the impact of the body’s metabolism of drugs became essential for identifying the therapeutically active moiety, as well as possible ‘toxic’ metabolites.The rise of chromatographyThe development of chromatographic techniques such as paper chromatography took place in the 1940s and allowed separation of the drug from its metabolites. Later in the 1950s, thin-layer chromatography was developed and used to quantify drugs in biological fluids, although its main application was in the separation of radiolabeled metabolites. Unfortunately, the sensitivity of these technologies was not sufficient to measure the new drugs of the 1950s, such as ‘tricyclics’ that had levels of ng/ml.In the early 1950s, gas chromatography (GC) became widely used in the pharmaceutical industry. The technique improved in sensitivity and selectivity with the development of more sensitive detectors, such as the flame ionization detector, which was modified to become the alkali flame ionization detector, alternatively named the nitrogen phosphorus detector with increased sensitivity for nitrogen- and phosphorus-containing compounds. The electron capture detector improved sensitivity even further for electroactive compounds. Further improvements in sensitivity and resolution came with the move from packed columns to capillary columns. Later, a wide variety of additional detectors became commercially available; for example, mass spectrometry, the widely used mass selective detector and a variety of special detectors, such as flame photometric, enhanced the scope of this technique. A major issue with GC was the almost inevitable need for complex sample preparation followed by derivitization, as GC requires the analyte to be volatile and thermostable.The ascendency of high-pressure liquid chromatographyIn the late 1960s and early 1970s, high-pressure liquid chromatography (HPLC) started to make in-roads; unlike GC, it did not require extensive sample preparation, derivitization or suffer from problems caused by thermal lability or the presence of water in the sample. HPLC increased in popularity as the range of detectors increased, allowing the technique to become more and more sensitive, in line with significant increases in the potency of therapeutic agents seen in the 1970s and 1980s. The earliest detectors were ultraviolet detectors; fluorescence detectors developed later were, for the ‘right’ compound, inherently more sensitive. Numerous postcolumn devices were developed to improve the native fluorescence of the analyte, including postcolumn irradiation with ultraviolet light and postcolumn reaction devices. Electrochemical detectors developed in the 1970s and 1980s had inherently high sensitivity; however, they were temperamental, required a high level of sample cleanliness and were not particularly robust.With the ever increasing need for a sensitive universal detector, it was not surprising that the Sciex atmospheric pressure interface between liquid chromatography and the mass spectrometer (HPLC–MS) became popular. As with all techniques, this too spawned a range of variants and improvements, most notably electrospray ionization and atmospheric pressure chemical ionization sources. This invention developed in the late 1980s, resulted in a technology that was universally accepted in a very short time. By the mid 1990s, this was the analytical tool of choice in bioanalysis, and saw the rapid decline in GC and HPLC methods with conventional detectors – today these techniques are reserved for esoteric assays.Electrophoretic technologiesWhile dramatic developments were taking place in HPLC, a range of electrophoretic-based technologies, starting with isotachopheresis in the mid 1970s, were being developed. Like conventional chromatography, these resulted in a range of techniques that have their own niche, especially capillary zone electrophoresis. This technique is restricted by the ability to apply only small volumes of samples which, when coupled with the short path lengths of the detection system, have limited sensitivity. Attempts to couple capillary zone electrophoresis with MS detectors although successful, have had limited application. These techniques showed high selectivity and great promise in measuring biologically related compounds, from peptides and oligonucleotides through to large peptides. Over the years, other related techniques such as micellar electrokinetic chromatography have been developed, each with their own idiosyncrasies.RadioimmunoassayThe quantitation of large molecules as therapeutic agents drove the development of ligand-based assays. This started with the development of a radioimmunoassay for the quantitation of insulin in the 1950s, while around the same time the thyroxine binding protein was used to measure thyroxine. Although the number of large molecular weight drugs was limited at this time, this technique offered the advantages of speed and sensitivity over some of the prevailing chromatographic techniques, such that it became the established technique for measuring some small molecules until the advent of liquid chromatography–tandem MS in the early 1990s. Ligand-based assays are now a major area of interest and it is expected that by 2010 30% of drugs in development will be biologicals; therefore, ligand- and cell-based assays are likely to grow in number and importance.Enzyme-mediated immunoassayAs the use of radioactivity fell in popularity, owing to handling and disposal issues, a wide variety of new immunoassay formats evolved. One of the earliest techniques was the enzyme-multiplied immunoassay technique, which was largely designed to measure drugs of abuse in urine in the late 1970s, although its application has now spread into the field of therapeutic drug monitoring. The radioactive signal was to a large extent replaced by an enzyme-mediated signaling system. The enzyme-multiplied immunoassay technique was a homogeneous immunoassay technique, the development of enzyme-linked immunosorbent techniques formed the basis of many new platforms, where the ligand (analyte) could be labeled with a different probe (e.g., fluorescent probe for fluorescence immunoassay). A further variant is the development of immunometric assays, where the antibody rather than the analyte (ligand) is labeled.Increased throughputThere is an ever-increasing demand upon analysts in general, and those within the pharmaceutical industry in particular, to produce more results faster and, as drug potency increases, using more sensitive techniques. Most recently, the field of ligand-based assays has seen the introduction of multiplexing assays (i.e., simultaneous quantitation of multiple analytes in the same sample using luminescence or chemiluminescence), which have expanded the capability of this technology and have been especially beneficial in servicing the need to quantitate multiple biomarkers. Owing to their enhanced sensitivity as a result of low background noise, they are being widely used for drug analysis, as well as measuring neutralizing antibodies. Flow cytometry has found wide use in multiplexed bead assays similar to those described. This technology has also been used in quantifying biomarkers and is growing in popularity.Miniaturization & nanotechnologiesAlthough this technology has not significantly impacted the field of bioanalysis, the use of microfuidic chips has found application in the quantitation of certain anions and cations in clinical chemistry. Chip-based capillary zone electrophoresis columns have helped minimize the inherent variability in this technique. On the chromatographic front, the need for more sensitive and faster assays has driven the development of an ultra high-pressure/performance chromatography using columns with 2 µm packing. This produces highly efficient columns, reducing assay time by approximately 70%. However, the concomitantly high back pressures require high pressure pumps and/or high temperatures to reduce the viscosity of the mobile phase.In immunoassays, the use of microfluidics, as used in the automated Gyros system, has increased sample throughput, reduced sample volume and improved the ease and speed of carrying out these types of assays many-fold. These new technologies are the forerunners of the emerging technologies that will become commonplace in the coming years.Niche technologies or the start of something big?Despite the decline in the use of radiolabels, it is still possible to use extremely low levels that can be dosed to humans so that absorption and, to some extent, metabolism can be studied using what has become termed ‘microdosing’. Accelerator mass spectrometry is used to quantify trace amounts of radiolabeled therapeutic molecules – specificity is maintained through chromatographic separation, the analyte is oxidized to carbon, which is then quantified by accelerator mass spectrometry. Although a laborious technique, it has found its niche in troubleshooting drug development issues.The cutting edge of analysis is based around the ability to identify, quantify and locate analytes at the cellular level. One of the most exciting aspects of in situ bioanalysis is the extension of immunohistochemical techniques, which in themselves can be semiquantitative, to the field of laser-ablation inductively coupled plasma mass spectrometry, which can map protein distribution using antibodies labeled with rare earth elements to target the protein(s) of interest.Regulatory issuesWhile the development of technologies in bioanalysis carried on at a pace, the actual criteria for characterizing the analytical method and ensuring it was under control varied from laboratory to laboratory. Many laboratories used tools from clinical chemistry or other analytical environments. However, there were no standard ways of doing this. One of the first formal regulatory-based guidances for carrying out bioanalysis was published in 1977 by the US FDA as part of the “Bioequivalence and Bioavailability” regulations.Since that time, both the FDA and the need for definitive bioanalytical criteria to be used in the measurement of drugs have driven the regulatory process. These bioanalytical criteria were discussed at the meeting “Analytical Methods Validation, Bioavailability, Bioequivalence and Pharmacokinetic Studies” in Crystal City, Arlington, VA, USA, 3–5 December 1990. This resulted in a conference report in 1992 and a subsequent “Draft Guidance for Industry: Bioanalytical Method Validation” in 1999. A meeting “Bioanalytical Methods Validation – a revisit with a decade of progress”, sponsored by the American association of Pharmaceutical Scientists and FDA, held in Arlington, VA, USA, 12–14 January 2000, discussed this draft guidance and resulted in a further conference report in the same year followed by the issue of a final FDA “Guidance for Industry on Bioanalytical Method Validation” in 2001. This guidance was developed largely for the quantitation of small molecules using chromatographic techniques, but now applies equally to large molecules and a variety of analytical techniques.Sample handlingAll biological samples contain extraneous material that can adversely impact many, if not all, of the analytical techniques described. Early attempts at ‘sample clean-up’ used organic solvents of varying polarity to extract the analyte followed by evaporation of the organic solvent. In the late 1970s and 1980s small plastic cartridges containing a solid phase capable of adsorbing and releasing the analyte under appropriate conditions were developed. In the 1990s, these were miniaturized to 96-well plate formats and above. In this format the use of automated and robotic handling systems became more routine and is still evolving today. These 96-well format systems, be they for sample preparation or routine processing of enzyme-linked immunosorbent assay plates, only become cost–effective where large numbers of samples are used. For smaller sample numbers the traditional liquid–liquid extraction and solid-phase extraction cartridges are still widely used.The use of 96-well plates also meant that reduced volumes of biological fluids could be assayed, allowing more sampling points to be taken and increased numbers of different assays to be carried out on the same sample.Recently, a method of spotting blood drops onto absorbent card, a technique borrowed from the clinical chemistry of pediatric samples, has become widely used, especially for species with small blood volumes. Whether this will gain universal acceptance remains to be seen and depends on the reproducibility of extracting the analyte of interest from the card.Data handlingAs the complexity of the analytical methods increased so has the need to handle more and more data. Only 40 years ago chromatographers were cutting out peaks and weighing them to measure peak areas, while rulers were used to define baselines and measure peak heights. It was not until the advent of electronic integrators in the late 1960s that measurement of peaks moved out of the manual and mechanical world. The 1970s saw the introduction of microprocessor integrators – the forerunners of the current computer-based systems that pervade all laboratories. Not only do these systems measure the instrument output, they generate standard curves from the simple to the complex and are able to generate concentration-related results, together with a plethora of parameters that can be used to evaluate the methodologies.The manipulation of these data into meaningful tabulated outputs of results and ‘supporting’ data, would not be possible in a timely manner without computers. How much of the data is generated because we can, rather than what is useful, remains a moot point. Unless it adds value it needs careful review. In spite of the large number of computer-based systems, the qualification, validation and interpretation of the outputs is still not as user friendly as it might be – developments continue as rapidly as the technologies they support. Ultimately, incorporation of the Electronic Laboratory Notebook with these data in a broad-based laboratory information management system remains the gold standard.Need for BioanalysisBioanalysis was a term derived in the 1970s to describe the process of quantifying drugs in biological fluids for the purposes of defining their pharmacokinetics. However, the techniques and technologies (mainly chromatographic- and ligand-based assays) cross many disciplines where drug analysis in biological fluids is important (e.g., forensic science, drugs of abuse and therapeutic drug monitoring). This is what this journal is all about.In recent years the term bioanalysis has been borrowed, or perhaps more generously seen as a parallel development, as a term used to define analytical techniques used in the quantification and characterization of biologicals. Many of these techniques and tools also find application in measuring biologicals in biological fluid – so the confusion continues.The boundaries between analysis of drugs for the purposes of pharmacokinetics, forensic science, biomarkers, clinical chemistry and therapeutic drug monitoring are becoming increasingly blurred. It is important to eliminate the silo mentality by cross fertilization of ideas between these different ‘disciplines’, while at the same time understanding the scientific and regulatory drivers behind them; a major objective of this journal is to catalyze this process.Once an analytical method was seen as an art form and the ability to be the person to get it to work seen as a unique skill, but no longer. Methods must be robust and able to be run by anybody with the appropriate training using highly automated systems; however, when things go wrong, a basic understanding of the equipment and processes are essential components of a bioanalyst’s repertoire. This journal should enhance the bioanalyst’s ability to troubleshoot by encouraging and sponsoring debate on ‘how to’ and fostering the development of best practices at both the technical and regulatory level.It is important to develop an understanding between the chemistry-based sciences and those that are biologically based in order to develop fit-for-purpose criteria relevant to the technology and its intended use.Bioanalysis will share and disseminate information in the conventional way by publishing novel manuscripts and cutting-edge reviews through the exchange of ideas and philosophies in an open forum. This can only work if the readers and the community it serves at large, respond in an engaged and constructive dialogue – it is hoped that Bioanalysis will provide this focus.Financial & competing interests disclosureThe author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.Bibliography1 Kanu AB, Hill HH. Ion mobility spectrometry detection for gas chromatography. J. Chromatogr. A1177(1),12–27 (2008).Crossref, Medline, CAS, Google Scholar2 Norman Dyson. Chromatographic Integration Methods. Royal Society of Chemistry, Cambridge, UK (1996).Google Scholar3 Joseph Chamberlain. 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This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.PDF download