Microsampling as the name implies, a very small amount of the matrix of organisms from animals and humans to determine drug concentration in bioanalysis. Microsampling has recently gained a lot of support Attention, which has led to the emergence of guidelines such as ICH, SA3, S11, and M10. The benefits of microsampling will help in the field of toxicokinetics, Pharmacokinetic tests, clinical trials, neonate samples, and remote sampling. Microsampling is strongly recommended from the 3R viewing area (Replace, Refine, and educe) in sample construction. Dry Blood Spot sampling is a type of Microsampling sample mentioned in this article in controlled bioanalysis with respect to bioanalytical considerations and process validation. A bioanalytical method is a set of processes involved in the collection, processing, storage, and analysis of a biological matrix of chemical compounds. Bioanalytical methodology (BMV) is a process used to determine whether a quantitative analysis method is suitable for chemical applications(Chapman et al. 2014). In the analysis of bioavailability findings, studies of bioequivalence (BE) pharmacokinetics (PK), drug screening tests, the development of new drugs, research in basic medical and pharmacological sciences, are the most important of these methods.
Guidelines for the validation of the bioanalytic method were also widely published by the FDA in 2001 and most recently by the EMEA. Through the use of unique laboratory investigations, verification includes documentation that system performance features are sufficient and accurate for targeted bioanalytical applications. The validity of the analysis information is consistent with the validation of the system used. Bioanalytical methods can be fully validated in critical studies that require regulatory action to be approved, such as BE or PK studies. A little verification can suffice for the high-level methods used to make internal sponsor decisions where a pre-certified approach can be required. There are always several changes; these changes should ensure the effective implementation of the analysis method. Demonstrating the legitimacy of the timely bioanalytical validation process: During the development and implementation of a new bioanalytic method, various changes are required to support specific studies of different levels of validation. Exploring a new drug. A review of the existing method increases the balance of metabolites.
Quantification of drug concentration is the basis of drug discovery and development. Its accuracy is a very important aspect of preclinical and clinical studies, therapeutic drug monitoring, and diagnostics. There are a wide range of matrix being used for sampling such as blood, plasma, serum, urine, saliva, milk, cerebral spine fluid, etc(Chapman et al. 2014). Routinely used for analyte concentration are plasma and serum, where the blood is withdrawn by a conventional venous collection method with the sample volume ranging from 500uL to 5mL. This standard method is considered invasive and also presents many drawbacks, besides it requires qualified personal for the sample collection and specific storage conditions and sometimes can even be time-critical. Controlled shipment and the large volume of samples are a few other issues that encouraged the development of alternative sampling techniques over the past few decades. Meanwhile, advanced LC-MS techniques became more affordable, allowing precise quantification in matrices, even with broad interfering compounds. Consequently, analytical challenges with whole blood were reduced to a minimum (Enderle, et al., 2016). Therefore, the interest and development of the use of micro-sampling of whole blood, for quantitative bioanalysis, has been growing in the past decades, making the blood plasma not necessarily the best matrix of choice to quantify therapeutic drug concentrations. Until then, the whole blood was not considered an efficient sample of choice as the rupture of Erythrocytes causing hemolysis would affect sample separation, management, and analysis.
The novel sampling technique with reduced blood sample volume emerged in order to overcome the above-mentioned drawbacks and is gaining major acceptance in the bioanalysis industry mainly due to the very reduced cost of the entire sampling process (from self-sampling to shipping and storage), the ethical treatment to humans and animals and regulatory compliance. To date, four microsampling techniques have been commercialized; Dried Blood Spot (DBS), Dried Plasma Spot (DPS), Capillary Micro Sampling (CMS) and Volumetric Absorptive Microsampling (VAMS).
The DBS technique involves a drop of whole blood volume in a filter card/paper and was introduced in the 60s for the screening of metabolic disorders in newborns (Kok and Fillet, 2018). Dried blood spot testing (DBS) is a form of bio sampling where blood samples are blotted and dried on filter paper. The dried samples can easily be shipped to an analytical laboratory and analyzed using various methods such as DNA amplification or HPLC. The collection of DBS samples is normally conducted by pricking the finger, heel, or toe with a lancet, and the blood drops are then spotted onto preprinted circles, ideally one drop per spot, on specially manufactured paper. Touching the circle area should be avoided, especially before the blood is applied and has completely dried. The blood sample can also be applied with a calibrated pipette onto the sampling paper, thus avoiding potential sampling errors. It is very important to dry blood spots completely before storage or transportation. In general, a minimum of 2–3 h drying in an open space at room temperature is recommended. However, the drying time depends on the type of paper and the blood volume applied. Thus, after drying, the DBS samples should be protected against humidity and moisture. Humidity indicator cards should be included in the storage package(Cohen et al. 2014; Jones et al. 2007). DBS samples protected in this manner may be stored at room temperature for many weeks, months, or years, depending on the analyte stability. Nevertheless, samples that contain unstable compounds should be stored at a lower temperature to enhance stability. DBS is a useful tool in forensic toxicology, but it has not replaced conventional whole blood, plasma, or serum samples. Although DBS offers several advantages over conventional matrices.
The collection area (finger, heel) has to be first disinfected. The skin is then punctured with a sterile lancet. The first blood drop is dabbed and subsequent drops are placed on blotting paper marked with circles to be filled. Once all the required circles are filled, the blotting paper is left to dry for a few hours at room temperature on a nonabsorbent surface. The drying time is very important as residual humidity favors bacterial development or molds and modifies the extraction stage. Conservation Once dry, the DBS cards are moved into a waterproof plastic bag, possibly along with a desiccant and a humidity indicator. The purpose of the desiccant is to finalize the drying process, which also minimizes any risk of infection associated with sampling. Periods of storage at room temperature vary according to the biological factor, from one week for proteins , to one year or more for nucleic acids . As far as serology is concerned, the blotting papers are usually kept at 20°C upon receipt . For long term preservation (up to several years) the blotting papers are stored either at 20°C or 80°C . Extraction
Extraction of the analytes from DBS specimens needs to be achieved using a standard procedure. One or more 2 to 8 mm diameter discs are then created with a specific punch. These small “spots” are placed in an elution buffer for variable time spans according to the procedure. The DBS extraction is then treated as a hemolyzed whole blood sample and tested with methods often intended for plasma or serum. The elution buffer plays a major role in stabalizibg the analytes to be tested. A wide variety of buffers are described in the literature. The most common are saline/phosphate buffers, often with added detergents, carrier proteins and (EDTA), as well as organic buffers with methanol, acetonitrile, or ethanol. For nucleic acids, standard commercial kits exist which are compatible with molecular biology tests, from PCR to genomic chips.
1.11 Primary sample preparation
The punch Sample preparation usually starts with the deportation of a segment of the DBS from the blotter using a manual or automated puncher. Commonly, to minimize the assay bias due to punch location, it is recommended to consistently take the DBS punch either from the center or close to the outer edge. The punch size may vary from 3 – 6 mm to the whole spot, depending on the method(Gonzalez C, et al.2000). Techniques have been developed to overcome the variations in hematocrit and also minimize the labor associated with the sample preparation process. Strategies to overcome the hematocrit effect include:
Pre-cutting or perforating the filter paper as part of the DBS handling procedure to recover the hematocrit effect and eliminate the chance of carry-over between the punches; Blotting of less whole blood volume (e.g. 10 µl) on the smaller pre-cut disk (3 or 6 mm) and analysis of the whole disk to disregard the hematocrit effect and improve the assay bias. A two-layered polymeric membrane to form a separated secondary dried plasma spot from the whole blood sample to be analyzed following solid-phase extraction ;
Development of a novel collection card for DBS sampling, which generates a volumetric plasma sample (2.5 or 5.0 µL) from a non-volumetric application of a whole blood sample. The purported advantages of this collection matrix include enhanced assay reproducibility and selectivity, with a simplified sample extraction procedure and elimination of the hematocrit effect.
Elution For analysis, the analyte of interest firstly requires elution out of the filter paper along with the whole blood matrix by using appropriate extractor buffers. The efficient elution of analytes from the DBS is challenging and there is always a chance of analyte loss due to ineffective extraction; poor sample elution is due to either incomplete extraction or analyte degradation. Hence, the choice of optimal extractor materials may vary from one compound to the other. As an example, pure methanol is considered a generic solvent for extraction of drugs of the blood spot sample. Water on the other hand impairs the interaction between cellulose and the target analyte’s hydroxyl groups and the partial addition of water before the organic extraction advances the efficiency in certain cases (eg. antivirals). To achieve effective analyte recovery with maximum extraction efficiency, the extraction parameters, including extractor solution mix, duration, temperature, and application of additional solvation energy (sonication), need to be optimized for each target metabolite.
1.13 Sample pre-treatment
A variety of sample preparation approaches have been suggested, with selection depending on the molecular characterization of the target compound. Incorporation of sample pre-treatment methods, either in combination with each other or in isolation, includes the classic sample preparation process of protein precipitation, liquid-liquid extraction (LLE); solid-phase extraction (SPE); supported liquid extraction (SLE); and/or derivatization.
1.14 Extraction and derivatization
Extraction and derivatization procedures applied manually (or offline) are considerably time-consuming and laborious. Whilst derivatization is not required for many plasma-based analytes using LC-MS/MS, it is required for many DBS analyses to improve the sensitivity; offsetting the small sample volume. However, as the derivatization process prolongs the overall analysis time it is considered to be a limiting factor and has been a driver for the development of on-line extraction techniques to facilitate the DBS sample pre-analytical treatment. Automation of sample preparation directly coupled with the LC-MS/MS system has been introduced to improve turn-around time and run cost. PPT is a simple and popular method for automation that has been utilized for TDM. However, following a single PPT procedure, salts and other endogenous analytes are still present which may cause ion suppression in the MS process. SPE-LC-MS/MS set-up is designed to facilitate online sample desorption and is a time and cost-effective method for DBS analysis. Compared to PPT, SPE presents an improved sample clean-up. There are specific challenges with on-line extraction approaches, in comparison with the off-line extraction methods which may be a significant source of assay bias, including; a non-homogenous mixture of internal standard (ISTD) with the analyte in the extract; sample dilution then bands broadening in chromatography separation; and/or inadequate focusing of the extract onto the analytical column (Gonzalez C, et al.2000). Accordingly, as part of the method development process, certain strategies are required to eliminate these issues.
Technology has been developed that allows for the direct sampling of the DBS, without the need for a change to liquid or elution. As it is described by the manufacturer, “Liquid Microjunction Surface Sampling Probes (LMJ-SSP) are self-aspirating devices where the liquid is pumped to and aspirated away from a surface of interest to a mass spectrometer for integrated extraction and ionization”. By utilizing the LMJ-SSP technology, the analyte of interest could be directly extracted from the different surfaces and detected by a mass spectrometer in a short time frame with minimal sample handling. The LMJ-SSP device coupled with the MS has been utilized for the determination of proteins in the DBS sample, direct tandem mass spectrometer for detection of hemoglobin, as well as therapeutic drugs. Likewise, a novel “on spot” direct derivatization approach provides a time and cost-effective alternative sample preparation procedure; a technique introduced to determine thiorphan drug.