Quantitative analysis of xenobiotics and endogenous compounds in biological samples using (ultra) high performance liquid chromatography [(U)HPLC] is essential in many disciplines of analytical science, from early-stage drug development through to clinical diagnostics, therapeutic drug monitoring (TDM), the anti-doping industry and forensic applications. These targeted quantitative bioanalyses are often conducted at the rate of a few minutes per injection (typically 10–20 samples per hour). However, in recent years, the use of short ‘guard’ columns (i.e., < 50 mm in length) fitted close to, or even directly to, a mass spectrometer (MS) ionisation source, coupled with high (U)HPLC flow rates has permitted significantly reduced analysis times of 30–60 s or less per sample, which can allow analysis of 96-well plates in less than one hour [1], [2], [3]. Fig. 1 shows an example of a TDM analysis for the anti-arrhythmic drug flecainide in human plasma which was reduced from an analysis time of 4 min to 1 min.

Whilst some applications of ‘rapid’ analysis, such as reaction monitoring in synthetic chemistry and quantitative purity investigations of contaminants in water are not directly related to the analysis of extracted biological samples, the benefits of such dramatically reduced analysis times would be transferrable to a number of application areas (e.g. oil and gas industry, environmental protection industry) in analytical science [4], [5], [6]. For targeted quantitative analyses in clinical diagnostics and TDM, rapid turnaround times would enable faster clinical decision-making and in drug discovery and drug development, high-throughput analysis allows faster screening of candidate compounds, and/or quicker understanding of pharmacokinetic parameters for both in vitro and in vivo studies [1,2].

An alternative approach to increasing throughput is to remove the chromatographic column entirely, and either use flow-injection analysis (FIA), i.e. introduction of a sample directly to the MS in a carrier solvent, or one of the many available ambient ionisation MS approaches [e.g. paper-spray MS, direct analysis in real-time (DART), laser diode thermal desorption (LDTD), Atmospheric Solids Analysis Probe (ASAP®), and others] [7], [8], [9], [10], [11]. There are also specialised instruments which use ultra-rapid flow-switching with solid phase extraction (SPE) cartridges and direct elution into the MS (RapidFire™ - Agilent technologies [12]). Undoubtedly, such approaches have been shown to be fit-for-purpose for some methods such as identification of new psychoactive substances and lipid metabolite changes and are in routine operation for high-throughput bioanalytical applications [13,14]. For others however, including a chromatographic separation step enables routine implementation. This may be due to known interferences which cannot be distinguished by the detector, e.g., isobaric species when using MS, or compounds with similar absorbance when using ultra-violet (UV) detection. For MS detection specifically, it is important to consider that structurally related compounds which are not isobaric (e.g. phase I and phase II conjugated metabolites) can become isobaric with parent drug ions due to in-source reactions/fragmentation [15]. Furthermore, in both low and high-resolution instrumentation, significant problems can arise due to ionisation effects from other co-eluting matrix components, as well as (U)HPLC eluent buffers and salts, etc. Matrix-related ionisation effects are especially problematic in bioanalysis given the highly complex sample matrices and often low concentration of target analytes combined with non-selective sample preparation techniques. Where chromatographic resolution becomes critical to the success of a bioanalytical method, but high throughput remains a pre-requisite, clearly the speed of the chromatographic step becomes all-important [16], [17], [18].

To date, the aforementioned applications of ultra-rapid chromatographic methods have been limited to one or more of the following factors: (i) relatively high-concentration target analytes, (ii) analytes for which stable isotopically-labelled internal standards (IS) are available, (iii) a small number of target analytes (rather than extensive analyte panels) or (iv) assays which involve extensive and/or selective sample preparation procedures to provide sufficiently ‘clean’ sample extracts. For MS methods, these limitations are likely due to factors including the effect of (U)HPLC eluent composition and flow rate on MS response, along with the complexity of the sample extracts (i.e., the amount of remaining sample matrix, and subsequent ionisation effects). Recent developments of reduced column length, i.e. < 5 cm (‘guard’ columns), can result in sufficient peak capacity for the separation of interferences, including matrix components and other compounds of interest [2,19].

With regards to the existing applications of rapid (U)HPLC, and their known limitations when considering broader applicability, we aimed to review the existing literature on the use of ‘rapid’ (U)HPLC methods to determine the current ‘state-of-play’ in approaches to bioanalytical applications. ‘Rapid’ is of course a subjective term; what is considered rapid for one application may be considered slow in other areas, but we aimed to use the literature as an observational approach to determine any relationships between various analytical and pre-analytical parameters such as, flow rate, internal standard (usage and chemistry), column chemistry and dimensional formats, matrix type and sample preparation techniques and overall ‘on-instrument’ analysis time (i.e. not including the time spent on sample preparation). Whilst some of these relationships are predicted by chromatographic theory, we aimed to see whether these theoretical considerations are being applied practically in the field. This is intended to give application scientists and researchers some insight into optimising analytical and pre-analytical parameter(s) to maximise assay throughput without compromising data quality.