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New Microfluidic-Based Sampling Procedure for Overcoming the Hematocrit Problem Associated with Dried Blood Spot Analysis
... The HemaXis DB, developed by Leuthold and colleagues, samples a fixed amount of blood onto regular DBS paper [55]. This device contains four capillaries with a fixed volume of 5.5 μl or 10 μl and a DBS filter which is slid into the device. ...
... The blood sample is then transferred onto the paper, which can be used for analysis. The Hct effect on the accuracy and precision of Mavoglurant quantification in the Hct range of 26-62% has shown to be within the acceptance criteria of ≤15% [55]. Compared with the HemaPEN device, the HemaXis maintains the classic DBS paper, which can be subsequently easily implemented in automated systems used for DBS analysis [57]. ...
... C American Chemical Society 2018. (C) Reprinted with permission from[55] (https://pubs.acs.org/doi/10.1021/ac503931g) C American Chemical Society (2015). ...
Over the last years, dried blood spot (DBS) sampling has gained significant interest due to development of analytical techniques combined with DBS, the simplicity and low cost of the method. Despite its wide use, DBS sampling can lead to inaccurate results due to the impact of the hematocrit (Hct) on the analysis. Some analytes have shown to be hardly impacted by Hct values. However, in other cases, a significant impact of Hct is observed, which requires the use of alternative approaches to circumvent this issue. This review describes the possible impact of Hct-related bias in DBS sampling in the context of hormone analysis and discusses the different methodologies that can be used to overcome this bias to ensure accurate results.
... There is an increasing interest in accurate volume DBS analysis as it eliminates the HCT-based area (volume) bias. Recently, new blood collection technologies such as volumetric absorptive microsampling (VAMS™) applied in Mitra™, microfluidic-based sampling applied in hemaPEN™ and HemaXis™, and the gravimetric method applied in volumetric DBS card are available for accurate volume blood microsampling [9][10][11][12][13][14][15] which has increased the potential of DBS samples in quantitative bioanalysis. Regardless of the sampling method, manual blood sampling triggers a physiological stress response [16] and can be a problem in some patients, in other words, patients with medical conditions leading to bruises, especially upon repeated skin pricks for serial blood sampling [17,18]. ...
... The mean blood volume calculated from cortisol concentrations in DBS samples spotted by these two methods differed by 3% with a similar level of precision (%CV, <3%). This variation in blood volume spotting was in agreement with other accurate volume DBS sampling systems [10,14,15]. All these results supported the delivery by Fluispotter of accurate and precise sample blood volumes in the form of DBS. ...
... future science group10.4155/bio-2020-0048 ...
Aim: A novel automated serial dried blood spot (DBS) sampler, ‘Fluispotter’, was tested for its sampling performance. Materials & methods: An LC–MS/MS method was developed for the analysis of cortisol in DBS samples serially spotted by Fluispotter. The cortisol concentrations in 148 paired DBS and plasma samples were compared across a hematocrit (HCT) range of 22–55%. Results: The interassay accuracy and precision were <10%. Overall assay bias was negligible across the HCTs tested when analyzing the whole-spot DBS samples. The accuracy and precision of the blood volume in 10 μl DBS samples spotted by Fluispotters and micropipettes were within 3%. Deming regression and Bland-Altman analysis showed a good agreement of DBS-predicted and measured plasma cortisol. Conclusion: The Fluispotter performed serial sampling with high accuracy and precision of the sample blood volume.
... Various approaches have been put forth and investigated in the literature to address the Ht-dependent blood spreading on paper substrates [10,16,17]. Such strategies include, among others, the application of techniques such as volumetric absorptive microsampling (VAMS) [18], perforated DBSs (PDBSs) [19], the employment of microfluidic-based sampling [20,21], an internal standard addition on the paper [22], as well as the application of dried plasma spots (DPSs) rather than DBSs [23,24]. Two newer methodologies for DBS The previously mentioned Ht effect contributes to the measurement bias of the analytical method, partly due to the fact that many DBS techniques use a fixed-diameter puncher (a partial punch of the spot); more blood is present in a fixed-size disc punched from a high-Ht (smaller) spot compared to the same originating from a low-Ht (larger) spot. ...
... Various approaches have been put forth and investigated in the literature to address the Ht-dependent blood spreading on paper substrates [10,16,17]. Such strategies include, among others, the application of techniques such as volumetric absorptive microsampling (VAMS) [18], perforated DBSs (PDBSs) [19], the employment of microfluidic-based sampling [20,21], an internal standard addition on the paper [22], as well as the application of dried plasma spots (DPSs) rather than DBSs [23,24]. Two newer methodologies for DBS sampling via "Ht-insensitive device" have been introduced more recently, namely volumetric absorptive paper disc (VAPD) and mini-disc (VAPDmini) [25]. ...
Dried blood spots (DBSs) are formed by collecting a small sample of blood on specialized filter paper and allowing it to dry naturally. Various domains of life sciences and drug research extensively use DBSs as a sampling technique. The “Hematocrit (Ht) effect” affects assay bias, and several strategies have been put forth to deal with it, including the correction of quantified concentrations using an appropriate correction factor. The approach was previously applied, following the utilization of an image processing algorithm developed in Matlab® to derive a reliable equation correlating DBS areas to Ht% values. The present work looks more closely at the application of image analysis to the evaluation of Ht in DBS samples. Utilizing image analysis software, DBS samples with known Ht values were processed. Preparation of cards has followed a previously developed protocol for the appropriate formation of uniform area DBSs, irrespective of Ht. The resulting areas showed close resemblance to the respective theoretical areas calculated by applying the correlation equation. Following that, the equation was utilized to determine the Ht values for each sample, and a comprehensive comparison of measured versus calculated Ht was carried out using various statistical approaches for method comparison. The results demonstrated a strong correlation, suggesting the method’s viability in estimating Ht for unknown DBS samples.
... HemaXis™ DB 10 contains a conventional DBS filter paper and four capillary channels with inlets for direct contact with a blood drop and outlets to indicate when a sufficient volume of blood is collected. Once the capillaries are filled, the DBS filter paper touches the outlets by the user manually closing the device and the accurate volume of blood (10 µL) is transferred to create DBS [83]. HemaSpot™ HF (SpotOn Sciences, San Francisco, CA, USA) has eight identical blade-shaped filter paper pieces arranged in a spiral to collect 9-10 µL of blood per blade. ...
... The swab material can absorb substantial sample volume, and the composition of the material can be engineered to achieve greater efficiency of release volume. Polyester and Rayon fiber swabs material can absorb significant amounts of sample volume but have minimal release volume (~33%) [84,85], while Nylon flocked swabs elute 97% of the sample off the swab very quickly with a~5-s vortex [83]. Swab collection devices are considered Class 1 devices by the FDA, and, thus, stringent requirements must be met for their composition and performance. ...
The ability to self-test for HIV is vital to preventing transmission, particularly when used in concert with HIV biomedical prevention modalities, such as pre-exposure prophylaxis (PrEP). In this paper, we review recent developments in HIV self-testing and self-sampling methods, and the potential future impact of novel materials and methods that emerged through efforts to develop more effective point-of-care (POC) SARS-CoV-2 diagnostics. We address the gaps in existing HIV self-testing technologies, where improvements in test sensitivity, sample-to-answer time, simplicity, and cost are needed to enhance diagnostic accuracy and widespread accessibility. We discuss potential paths toward the next generation of HIV self-testing through sample collection materials, biosensing assay techniques, and miniaturized instrumentation. We discuss the implications for other applications, such as self-monitoring of HIV viral load and other infectious diseases.
... A first alternative device is the Mitra R volumetric microsampling device (Neoteryx, USA) [ 113 , 114 ], which entered the market in 2014 [115] . Other examples of volumetric microsampling devices that have been marketed since then include the HemaXis DB10 device (DBS system SA, Switserland) [116] , the Capitainer R qDBS device (previously Capitainer R B) (KTH, Stockholm, Sweden) [ 117 , 118 ] and the hemaPEN R device (Trajan, Melbourne, Australia) [ 119 , 106 ] ( Fig. 1 ). As most of these devices allow volumetric collection of blood, analysis of the complete dried microsample overcomes the impact of the hct on the spreading of blood on filter paper. ...
... The HemaXis device is a plastic foldable microfluidic-based device, comprising of a standard DBS paper card and a microfluidic chip containing four capillaries. Following contact between the capillary inlet and a drop of blood, the capillary channel fills itself with a fixed amount of blood, as depicted in Fig. 1 , Panel B [116] . Following closure of the device, the outlet of the capillary channel makes contact with a conventional DBS card, thereby emptying the capillary's content (10 μL of blood) onto the card. ...
In the field of solid organ transplantation, chemotherapy and autoimmune disorders, treatment with immunosuppressant drugs requires intensive follow-up of the blood concentrations via therapeutic drug monitoring (TDM) because of their narrow therapeutic window and high intra- and inter-subject variability. This requires frequent hospital visits and venepunctures to allow the determination of these analytes, putting a high burden on the patients. In the context of patient-centric thinking, it is becoming increasingly established that at least part of these conventional blood draws could be replaced by microsampling, allowing home-sampling and increasing the quality of life for these patients. In this review we discuss the published methods - mostly using liquid chromatography coupled to tandem mass spectrometry - that have utilized (volumetric) dried blood samples as an alternative for conventional liquid whole blood for the TDM of immunosuppressant drugs. Furthermore, some pre-analytical considerations using DBS or volumetric alternatives are considered, as well as the applicability on clinical samples. The implementation status in clinical practice is also discussed, including (1) the cost-effectiveness of this approach compared to venepuncture, (2) the availability of multiplexed methods, (3) the status of harmonization and (4) patient perception. A brief perspective on potential future developments for the dried blood-based TDM of immunosuppressant drugs is provided, by considering how obstacles for the implementation of these strategies into clinical practice might be overcome.
... However, they concluded on the possible use of this method for the analysis of seven elements (Cu, Fe, K, Na, Mg, Se and Zn) but raised a thought on the necessity to consider hematocrit values. To overcome this issue, the use of accurate volume for DBS sampling and the punch of the whole DBS are recommended (2,9). The use of a microfluidic system in the sampling device as presented on HemaXis DB10 allows a precise volume control during DBS collection (9). ...
... To overcome this issue, the use of accurate volume for DBS sampling and the punch of the whole DBS are recommended (2,9). The use of a microfluidic system in the sampling device as presented on HemaXis DB10 allows a precise volume control during DBS collection (9). Although Parsons et al. highlighted in their review these problems of filter paper contamination, inadequate limits of detection and lack of well-validated field-based studies faced in various DBS biomonitoring studies, they concluded on the usefulness of this technique as a screening method in case of acute concentrations of Pb, Cd and Hg. ...
Microsampling techniques became more popular in the last decades and their use for common analysis such as trace elements quantification by inductively coupled plasma mass spectrometry (ICP-MS) has been investigated. We decided to compare two of these techniques (dried blood spots and microtubes) to evaluate their potential for the analysis of twelve trace elements in human whole blood: aluminum (Al), total arsenic (As), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), manganese (Mn), molybdenum (Mo), nickel (Ni), lead (Pb), selenium (Se) and zinc (Zn). Signal contributions from blank filter paper and instability at room temperature for several elements in the dried blood spot samples restrained our enthusiasm for the use of this technique. Conversely, microtube samples presented low background contamination and good stability under different temperature conditions. Therefore, our results demonstrate that the use of microtubes is more suitable than dried blood spots for trace elements quantification in human blood, both in research and routine analysis.
... Quality control (QC) samples, including the lower limit of quantification (LLOQ), low QC (LQC), medium QC (MQC), and high QC (HQC) were prepared from separate stock solutions in the same way as calibration standards at concentrations of 1,000, 3,000, 30,000, and 80,000 ng/mL for caffeine, while 10.0, 30.0, 300, and 800 ng/mL for paraxanthine, theobromine, and theophylline. Blank whole blood was adjusted to a hematocrit level of 40 % as described before [32], which corresponded to the mean hematocrit of preterm infants was used to prepare calibrators and QC samples. Calibration standards and quality control samples were freshly prepared before each analytical run, except for the stability validation. ...
Objectives:
To update traditional "wet" matrices to dried blood spot (DBS) sampling, based on the liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) technique, and develop a method for simultaneous analyzing caffeine and its three primary metabolites (theobromine, paraxanthine, and theophylline), supporting routine therapeutic drug monitoring (TDM) for preterm infants.
Methods:
DBS samples were prepared by a two-step quantitative sampling method, i.e., volumetric sampling of a quantitative 10 μL volume of peripheral blood and an 8 mm diameter whole punch extraction by a methanol/water (80/20, v/v) mixture containing 125 mM formic acid. Four paired stable isotope labeled internal standards and a collision energy defect strategy were applied for the method optimization. The method was fully validated following international guidelines and industrial recommendations on DBS analysis. Cross validation with previously developed plasma method was also proceeded. The validated method was then implemented on the TDM for preterm infants.
Results:
The two-step quantitative sampling strategy and a high recovery extraction method were developed and optimized. The method validation results were all within the acceptable criteria. Satisfactory parallelism, concordance, and correlation were observed between DBS and plasma concentrations of the four analytes. The method was applied to provide routine TDM services to 20 preterm infants.
Conclusions:
A versatile LC-MS/MS platform for simultaneous monitoring caffeine and its three primary metabolites was developed, fully validated, and successfully applied into the routine clinical TDM practices. Sampling method switching from "wet" matrices to "dry" DBS will facilitate and support the precision dosing of caffeine for preterm infants.
... 9 Several DBM techniques have been developed to overcome these limitations by facilitating the collection of volumetric samples with superior potential for sample integrity, including volumetric microsampling sampling (VAMS) tips (Mitra; Neoteryx, Torrance, CA and Tasso-M20; Tasso, Inc Seattle, WA) and the application of microfluidics (HemaSpot, Spot on Sciences (San Francisco, CA); HemaXis, DBS System SA (Gland, Switzerland); hemaPEN, Trajan Scientific and Medical (VIC, Australia); and Capitainer-B, Capitainer AB (Solna, Sweden)). [10][11][12][13][14][15][16][17][18][19] VAMS uses a porous, hydrophilic, polymeric tip to wick up a consistent fixed volume of 10, 20, or 30 mL by capillary action from a drop of capillary blood produced by a lancet. 10 Two commercially available devices currently use VAMS tips, namely, Mitra (Neoteryx, Torrance, CA) and Tasso-M20 (Tasso, Inc, Seattle, WA). ...
Sigma 2 receptor (σ2R) is overexpressed in select cancers and is regarded as a biomarker for tumor proliferation. σ2R ligands are emerging as promising theranostics for cancer and neurodegenerative diseases. Herein, we describe the design and synthesis of a series of novel quinolyl pyrazinamides as selective and potent σ2R ligands that show sub-micromolar potency in pancreatic cancer cell lines. Compounds 14 (JR1-157) and 17 (JR2-298) bind σ2R with Ki of 47 and 10 nM, respectively. Importantly, compound 14 has an oral bioavailability of 60% and shows significant in vivo efficacy without obvious toxicity in a syngeneic model of pancreatic cancer. The cytotoxicity of the quinolyl pyrazinamides significantly enhanced in the presence of copper and diminished in the presence of the copper-chelator tetrathiomolybdate. In conclusion, compound 14 is water-soluble, metabolically stable, orally active, and increases the expression of the autophagy marker LC3B and warrants further development for the treatment of pancreatic cancer.
... By disposing or collecting a fixed volume of blood, these devices allow to overcome the impact of the area bias. Examples of such devices include the Mitra® volumetric absorptive microsampling device (Neoteryx, Torrance, USA) [9,10], the HemaXis device (DBS system SA, Switzerland) [11], the HemaPEN® (Trajan, Melbourne, Australia) [12] and the Capitainer-B device (Capitainer AB, Solna, Sweden) [13,14]. The latter device recently received an 'upgrade' to yield the Capitainer® qDBS (quantitative Dried Blood Spot) device (Capitainer AB, Solna, Sweden) [15]. ...
In recent years, a lot of attention has been given to a more patient-centric therapeutic drug monitoring (TDM) of immunosuppressant drugs (tacrolimus, sirolimus, everolimus and cyclosporin A) by the use of microsampling techniques. By adopting Dried Blood Spots (DBS) after a finger prick, instead of conventional venous blood draws, follow-up can (partially) be established from patients' homes. Despite the many advantages of DBS, one of the major disadvantages associated with this technique is the well described hematocrit (hct) effect. In order to overcome the hct area bias, different strategies have been proposed, amongst which the use of dried blood sampling techniques based on the volumetric collection of blood. The aim of this study was to evaluate the use of the Capitainer® qDBS (quantitative Dried Blood Spot) device for the combined TDM of four immunosuppressants and creatinine from a single qDBS. The set-up of an adequate sample preparation allowing both immunosuppressants and creatinine quantification was one of the key challenges in the method development due to device-specific interferences. Liquid chromatography tandem-mass spectrometry methods for the quantification of tacrolimus, sirolimus, everolimus, cyclosporin A and creatinine from qDBS (10 μL) were developed and validated based on international guidelines, also taking into account DBS-specific parameters. The methods proved to be accurate and reproducible, with absolute biases below 10% and within-run CVs (%) below 8% over a calibration range from 1 to 50 ng/mL for tacrolimus, sirolimus and everolimus, 20-1500 ng/mL for cyclosporin A, and 15-700 μmol/L for creatinine. Reproducible (CV < 15%) IS-compensated relative recovery values were obtained, showing no hematocrit-dependence (compared to a hct of 0.37), except for cyclosporin A at higher hct values. Application on venous blood left-over patient samples showed good agreement between the results of Capitainer® qDBS and whole blood with 98% (47/48), 93% (41/44), 89% (41/46), 88% (38/43) and 89% (116/131) of the samples lying within 20% of the whole blood result for tacrolimus, sirolimus, everolimus, cyclosporin A and plasma/serum for creatinine, respectively. For creatinine a blood/plasma ratio of 0.85 was found and used to convert qDBS results to plasma/serum results. As a next step, capillary finger prick samples will need to demonstrate the clinical applicability of the method in a real life setting.
... The Hemaxis DB10 device comprises four fixed-volume capillary channels (e.g., 5 or 10 μl) and removable filter paper to generate blood spots without needing a pipette ( Figure 6c). 75,76 Blood is collected directly from a finger stick into each individual capillary. Closing the lid of the device brings the filter paper into contact with the capillaries to produce four replicate blood spots. ...
In the wake of the COVID‐19 global pandemic, self‐administered microsampling tools have reemerged as an effective means to maintain routine healthcare assessments without inundating hospitals or clinics. Finger‐stick collection of blood is easily performed at home, in the workplace, or at the point‐of‐care, obviating the need for a trained phlebotomist. While the initial collection of blood is facile, the diagnostic or clinical utility of the sample is dependent on how the sample is processed and stored prior to transport to an analytical laboratory. The past decade has seen incredible innovation for the development of new materials and technologies to collect low‐volume samples of blood with excellent precision that operate independently of the hematocrit effect. The final application of that blood (i.e., the test to be performed) ultimately dictates the collection and storage approach as certain materials or chemical reagents can render a sample diagnostically useless. Consequently, there is not a single microsampling tool that is capable of addressing every clinical need at this time. In this review, we highlight technologies designed for patient‐centric microsampling blood at the point‐of‐care and discuss their utility for quantitative sampling as a function of collection material and technique. In addition to surveying methods for collecting and storing whole blood, we emphasize the need for direct separation of the cellular and liquid components of blood to produce cell‐free plasma to expand clinical utility. Integrating advanced functionality while maintaining simple user operation presents a viable means of revolutionizing self‐administered microsampling, establishing new avenues for innovation in materials science, and expanding access to healthcare.
... Two book-style devices also integrate fixed-volume capillary channels to apply discrete volumes of blood to traditional DBS filter papers. The Hemaxis DB10 device comprises four fixedvolume capillary channels (e.g., 5 or 10 µL) and removable filter paper to generate blood spots without needing a pipette ( Figure 6C) [75]. Blood is collected directly from a fingerstick into each individual capillary. ...
In the wake of the COVID-19 global pandemic, self-administered microsampling tools have reemerged as an effective means to maintain routine healthcare assessments without inundating hospitals or clinics. Fingerstick collection of blood is easily performed at home, in the workplace, or at the point-of-care, obviating the need for a trained phlebotomist. While the initial collection of blood is facile, the diagnostic or clinical utility of the sample is dependent on how the sample is processed and stored prior to transport to an analytical laboratory. The past decade has seen incredible innovation for the development of new materials and technologies to collect low-volume samples of blood with excellent precision that operate independently of the hematocrit effect. The final application of that blood (i.e., the test to be performed) ultimately dictates the collection and storage approach as certain materials or chemical reagents can render a sample diagnostically useless. Consequently, there is not a single microsampling tool that is capable of addressing every clinical need at this time. In this review, we highlight technologies designed for patient-centric microsampling blood at the point-of-care and discuss their utility for quantitative sampling as a function of collection material and technique. In addition to surveying methods for collecting and storing whole blood, we emphasize the need for direct separation of the cellular and liquid components of blood to produce cell-free plasma to expand clinical utility. Integrating advanced functionality while maintaining simple user operation presents a viable means of revolutionizing self-administered microsampling, establishing new avenues for innovation in materials science, and expanding access to healthcare.
... In addition, to accommodate laboratories that use the HemaXis sampling technique, samples were prepared on 10 mL HemaXis sampling cards. 35 The weighed-in values can be found in Table 1. The Ht value of the used blood was 0.36 (v/v). ...
Background:
Therapeutic Drug Monitoring (TDM) of immunosuppressive drugs is important for the prevention of allograft rejection in transplant patients. Several hospitals offer a microsampling service that provides patients the opportunity to sample a drop of blood from a fingerprick at home that can then be sent to the laboratory by mail. The aim of this study was to pilot an external quality control program.
Methods:
Fourteen laboratories from seven countries participated (fully or partly) in three rounds of proficiency testing for the immunosuppressants tacrolimus, ciclosporin, everolimus, sirolimus and mycophenolic acid. The microsampling devices included the following: Whatman 903 and DMPK-C, HemaXis, Mitra and Capitainer-B. All assays were based on LC-MS/MS. In round 2, microsamples as well as liquid whole blood samples were sent, and one of these samples was a patient sample.
Results:
Imprecision CV% values for the tacrolimus microsamples reported by individual laboratories ranged from 13.2% - 18.2%, 11.7% - 16.3%, and 12.2% - 18.6% for rounds 1, 2, and 3 respectively. For liquid whole blood (round 2), the imprecision CV% values ranged from 3.9% - 4.9%. For the other immunosuppressants, the results were similar. A great variety in analytical procedures was observed, especially the extraction method. For the patient sample, the microsample results led to different clinical decisions compared to that of the whole blood sample.
Conclusions:
Immunosuppressant microsampling methods show great interlaboratory variation compared to whole blood methods. This variation can influence clinical decision making. Thus, harmonization and standardization are needed. Proficiency testing should be performed regularly for laboratories that use immunosuppressant microsampling techniques in patient care.
... The VAMS technology has been studied for the TDM of different classes of drugs such as antidepressants, antiepileptics, antibiotics, and analgesics, exploiting different biological matrices (25)(26)(27)(28). However, microfluidic-generated DBS (mfDBS) technologies have not been widely investigated for TDM purposes (29)(30)(31). ...
Clozapine is one of the most widely used second-generation antipsychotic drugs (SGAs) for the treatment of schizophrenia. Despite advantages over first-generation drugs, clozapine still shows significant side effects and interindividual variations in efficacy. In order to ensure frequent therapeutic drug monitoring (TDM) and improve the compliance of psychiatric patients undergoing clozapine treatment, two novel dried microsampling approaches based on whole blood and plasma volumetric absorptive microsampling (b-VAMS and p-VAMS) and microfluidic generated-dried blood spot technology (mfDBS) were developed and coupled to HPLC with electrochemical detection (ED). The proposed miniaturized strategies by means of VAMS and microfluidic channel-based devices provide several advantages in terms of collection, storage, and handling compared to classical blood and plasma processing. Satisfactory validation results were obtained for all microsampling platforms, with mean extraction yields >85.1%, precision as relative standard deviation (RSD) < 5.1%, and stability < 4.5% analyte loss after 30 days for p-VAMS; mean extraction yields > 83.4%, precision RSD < 5.4%, and stability < 4.6% analyte loss after 30 days for b-VAMS, and mean extraction yields > 74.0%, precision RSD < 5.6%, and stability < 4.9% analyte loss after 30 days for mfDBS. The original microsampling methodologies have been successfully applied to the blood and plasma collected from five psychiatric patients for the monitoring of the levels of clozapine and its main metabolites, providing robust and reliable quali-quantitative results. Comparisons between results of the two dried microsampling technologies with those obtained by classic fluid plasma analysis were in good agreement and have demonstrated that the proposed miniaturized approaches could be suitable for TDM purposes.
... A HemaXis auxiliary device (DBS System SA, Switzerland) for taking a fixed volume of blood integrated with a standard card from Whatman was presented [72]. This device makes it possible to apply blood in a fixed volume to the membrane at the center of a circle using a capillary system (Fig. 7). ...
... In clinical practice, a precise volumetric blood spot is difficult to obtain because microsampling devices are frequently used by untrained personnel or patients themselves at home. Different strategies have been recently proposed to overcome this limitation by developing various devices to generate a volumetric spot from a non-volumetric blood drop, such as HemaXis, hemaPEN and in situ generated DPS (36)(37)(38)(39). Alternatively, hematocrit correction strategies need to be used for non-volumetrically generated DBS after hematocrit evaluation on DBS samples by spectroscopic techniques (40). ...
Introduction: dose adjustment of antibiotic drugs is fundamental in critically ill patients and newborns, who may experience inadequate exposure of these drugs due to different pharmacokinetics (PK) and pharmacodynamics (PD) compared to other patients. Despite the increasing use of microsampling devices such as Dried Blood Spot (DBS) and Volumetric Absorptive Microsampling (VAMS) in paediatric clinical practice, little is reported on the comparison between these two devices in terms of β-lactam antibiotics quantification. Methods: a method using high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) for the simultaneous determination of amoxicillin, ampicillin, meropenem and cefadroxil from both DBS and VAMS has been evaluated. More specifically, we investigated the influence of some well-known critical pre-analytical parameters, such as blood haematocrit, blood spot volume and sample recovery approach on the results obtained with DBS-based and VAMS-based analytical methods. Results: our results show that comparable trueness and precision values can be achieved using both DBS-or VAMS-based methods. However, the latter was not affected by blood haematocrit value, whereas accuracy of the DBS-based approach was influenced by parameters as haematocrit, blood spot approaches (by pipette or hanging drop) and spot recovery procedures (DBS cut or punch). For the β-lactams tested, a significant longer stability was found for samples absorbed on DBS, when samples were stored at 4°C. Discussion: our data support the use of VAMS as preferable devices for β-lactam determinations, while more attention needs to be devoted to different pre-analytical variables for an accurate DBS quantification.
... Launched in 2014, volumetric absorptive microsampling (VAMS) is a fixed volume collection device that can be used to sample blood easily. A hydrophilic tip of VAMS absorbs a specific volume of blood through capillary action and analysis is performed after drying [7,8]. Within a few seconds of finger prick at a 45° angle at fingertips, the VAMS tip can directly contact the blood droplet, and a precise volume of blood (10 µL) is collected within the hydrophilic tip. ...
Acetylsalicylic acid (ASA) is one of the most commonly used medications in global market, with a risk of intoxication in certain patients. However, monitoring blood drug concentration often requires frequent hospital visits; hence there is an unmet need to increase patient-centricity by conducting blood sampling at home. Volumetric absorptive microsampling (VAMS) is a device that allows collection of homogenous and accurate volume of blood without venipuncture, and can be utilized by patients who are not in hospital settings; but because ASA is prone to hydrolysis and stabilizing reagents cannot be added to VAMS samples, a way to improve sample stability must be developed. The objective of this study was to identify the cause of instability with ASA samples collected by VAMS, and to evaluate ways to improve sample stability. A liquid chromatography with tandem mass spectrometry (LC-MS/MS) was used for analysis of ASA concentration in whole blood. Samples collected with VAMS were kept under different drying conditions (desiccator, pressurized, nitrogen gas and household vacuum sealer) and were compared to the control samples collected by conventional venous sampling. The recovery of ASA was about 31% of the control when VAMS sample was dried at room temperature, whereas VAMS samples under humidity controlled conditions showed more than 85% of recovery. Our results suggest that adequate level of humidity control was critical to ensure sample stability of ASA, and this humidity control could also be achieved at home using household vacuum sealer, thus enabling patient-centric clinical trials to be conducted.
... As a consequence, the DBS technology cannot be a general alternative for sampling in pharmacokinetic studies [24]. As for VAMS, it involves microvolumetric absorption and the subsequent drying of the liquid sample in a porous tip that can collect 10-20 μL blood for storage in cartridges, clamshells, or 96-well racks [25][26][27]. VAMS has been extensively used for pharmacokinetic studies and therapeutic drug monitor [28,29]. However, the cost of the special devices is high because of a large number is needed in pharmacokinetic studies. ...
Mice are the most frequently used animals in pharmacokinetic studies; however, collecting series of blood samples from mice is difficult because of their small sizes and tiny vessels. In addition, due to the small sample size, it is problematic to perform high required quantification. Thus, present work aims to find an effective strategy for overcoming these challenges using trans-resveratrol as a tool drug. Based on the idea of a joint technology, the capillary microsampling (CMS) was chosen for blood sample collection from mice after delivery of trans-resveratrol (150 mg/kg) by gavage, and a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for the determination of trans-resveratrol and its main metabolites. All the mouse blood samples were exactly collected by CMS without obvious deviation. This provided credible samples for subsequent quantitative analysis. The HPLC-MS/MS method was found to be sensitive, accurate, and repeatable, and the pharmacokinetic parameters for all analytes were comparable with those reported in previous studies. However, the present joint technology offers the advantages of less animal damage, easy for sample preparation, and improved reliability. It has overcome some of the major limitations revealed in previous pharmacokinetic studies in mice and therefore provides a more effective option for future studies.
... A new generation of microsampling devices has been proposed to extend the volumetric DBS application to untrained people and make self-sampling at home possible. HemaSpot TM (Spot on Science, San Francisco, CA) [35], volumetric absorptive microsampling (VAMS) (Neoteryx, USA [23,[36][37][38], volumetric absorptive paper disc (VAPD) [39], and microfluidic-based systems, as HemaXis DB (DBS System SA, Gland, Switzerland) [40,41] and Capitainer-B (Capitainer AB, Stockholm, Sweden) [26,[42][43][44], are just a few illustrative user-friendly devices on the market [45]. Even when the analysis of the whole spot is performed, the influence of HCT variability on the extraction efficiency should be considered [28]. ...
Dried Blood Spot (DBS) analysis is a well-established practice for monitoring newborns in neonatal units because of the non-invasive collection of a very limited blood volume. Unfortunately, the haematocrit level and the drop volume can adversely affect the quality of the analytical data. For this reason, we performed an in-depth comparison of non-volumetric and volumetric sampling (e.g. Minivette® POCT microcapillary, and the microfluidic-based systems HemaXisTM DB and Capitainer-B) for the DBS analysis of the isoprostanoids 8-iso prostaglandin F2α and 8-iso prostaglandin E2, and prostaglandin E2, which represent well recognized indexes of oxidative stress and inflammation.
Results highlighted a significant impact of drop volume on the analytical performances for the non-volumetric approach due to the inhomogeneous distribution of the analytes across the DBS. The innovative addition of labelled internal standards (ISs) on the filter paper before sampling did not circumvent the problem, as ISs did not accurately mimic the spreading of analytes in the DBS. Differently from HemaXisTM DB and Capitainer-B, the Minivette® POCT device showed an almost quantitative recovery with an overall variability of about 15%, and resulted the best option for an easy and reliable DBS collection in the clinic. Our procedure was used for the determination of the three metabolites in preterm newborns suffering from Patent Ductus Arteriosus (PDA). During our longitudinal monitoring, most of the preterm newborns with PDA showed a marked decrease (40-60%) of target analytes upon ibuprofen therapy, suggesting the potential involvement of the isoprostanoids and prostanoids in promoting drug responsiveness and ductal closure.
... 12,13 Due to the increased use of DBS and convenience for remote sampling, tremendous research and development have been undertaken to improve the consistency and analysis of DBS samples. 5, [14][15][16] However, a liquid blood sample can provide a greater volume of blood (>100 µL). An increased sample volume may be desirable for applications such as genomics, transcriptomics, or the detection of rare analytes. ...
Gene expression analysis (e.g., targeted small gene panels, transcriptomics) from whole blood can elucidate mechanisms of immune function and aid in the discovery of biomarkers. Conventional in-clinic venipuncture offers only a small snapshot of our broad immune landscape as immune responses may occur outside of the time and location parameters available for conventional venipuncture. A self-operated method that enables flexible sampling of liquid whole blood coupled with an immediate stabilization of cellular RNA is instrumental in facilitating capture and preservation of acute or transient immune fluxes. To this end, we developed home RNA: a kit that allows for self-collection of peripheral blood (~0.5 mL) and immediate stabilization of cellular RNA, using the Tasso-SST™ blood collection device paired with a specially designed stabilizer tube containing RNA later ™. To assess the usability and feasibility of home RNA for self-collection and stabilization of whole blood RNA, we conducted a pilot study ( n = 41 participants) where we sent homeRNA to participants aged 21-69, located across 10 US states (94% successful blood collections, n = 51). Among participants who successfully collected blood, 91% reported no or minimal pain/discomfort using the kit ( n = 35), and 77% reported easy or somewhat easy stabilization protocol. Total RNA yield from the stabilized samples ranged between 0.24 µg and 5.99 µg (mean = 1.65 µg), while RNA Integrity Number (RIN) values were above 7.0 (mean = 7.9), indicating limited RNA degradation. Results from this study demonstrate the self-collection and RNA stabilization of whole blood with home RNA by participants themselves, in their own home.
... After filling the capillary channel, the device was manually closed, allowing the outlet of the capillary to be in contact with the card, resulting in the transfer of a defined volume of blood (10 µL) to the DBS card, which takes about 5 s. [68][69][70] After completion of the transfer, the capillary channel should be completely empty, which can their own blood. The kit was classified as "very easy "or "easy" to use by 83.6% of participants who mailed the kit to the lab. ...
Dried blood spots (DBS) have been utilized in newborn screening programs for several years. More recently, there has been growing interest in using DBS as a home sampling tool for the quantitative determination of analytes. However, this presents challenges, mainly due to the well-known hematocrit effect and other DBS-specific parameters, including spotted volume and punch site, which could add to the method uncertainty. Therefore, new microsampling devices that quantitatively collect capillary dried blood are continuously being developed.In this review, we provided an overview of devices that are commercially available or under development that allow the quantitative (volumetric) collection of dried blood(-based) microsamples, and are meant to be used for home or remote sampling. Considering the field of therapeutic drug monitoring (TDM), we examined different aspects that are important for a device to be implemented in clinical practice, including ease of patient use, technical performance, and ease of integration in the workflow of a clinical laboratory. Costs related to microsampling devices are briefly discussed, as this additionally plays an important role in the decision-making process.Although the added-value of home sampling for TDM and the willingness of patients to perform home sampling has been demonstrated in some studies, real clinical implementation is progressing at a slower pace. More extensive evaluation of these newly developed devices, not only analytically but also clinically, is needed to demonstrate their real-life applicability, which is a prerequisite for their use in the field of TDM.
... In addition, CMS has been coupled with DBS to overcome the volume variability in the collection of blood samples. However, capillary devices also represent a promising approach for TDM in neonates [37]. As an example, in a comparative PK study on neonates, our group has successfully used microtubes to collect capillary blood samples using a heel stick, overcoming phlebotomy [38]. ...
Therapeutic drug monitoring (TDM) should be adopted in all neonatal intensive care units (NICUs), where the most preterm and fragile babies are hospitalized and treated with many drugs, considering that organs and metabolic pathways undergo deep and progressive maturation processes after birth. Different developmental changes are involved in interindividual variability in response to drugs. A crucial point of TDM is the choice of the bioanalytical method and of the sample to use. TDM in neonates is primarily used for antibiotics, antifungals, and antiepileptic drugs in clinical practice. TDM appears to be particularly promising in specific populations: neonates who undergo therapeutic hypothermia or extracorporeal life support, preterm infants, infants who need a tailored dose of anticancer drugs. This review provides an overview of the latest advances in this field, showing options for a personalized therapy in newborns and infants.
... In order to overcome these issues, other microsampling approaches closely related to DBS have been investigated such as dried plasma spots (DPS) and dried serum spots (DSS), as well as newer possibilities such as volumetric absorptive microsampling (VAMS) and microfluidic-based techniques. [10][11][12][13][14][15][16] A recently developed technology based on capillary volumetric blood microsampling (hemaPEN®) allows the simultaneous collection of four identical DBS replicates (2.74 μL each) from a single sample, thanks to the presence of four end-to-end EDTA-coated microcapillaries. The pen-like design of this device makes it easy to handle and, with the inclusion of a desiccant, sample integrity is ensured while preventing any contamination. ...
Therapeutic drug monitoring (TDM) is an important tool for correlating the administered drug dose to drug and metabolite concentrations in the body and to therapeutic and adverse effects. In the case of treatment with drugs active on the central nervous system (CNS), frequent TDM becomes really useful, especially for patient compliance checking and for therapy optimisation. The selective serotonin reuptake inhibitors (SSRIs) fluoxetine and sertraline, chosen as target compounds for this study, are two antidepressants mainly used for major depression, but also for obsessive-compulsive disorder associated with neurodegenerative diseases and for eating disorders. Microsampling approaches can be used to make TDM patient-friendly, by means of minimally invasive fingerpricking instead of classic invasive venipuncture. In this study, an innovative volumetric microsampling approach based on the use of hemaPEN technology is proposed to simultaneously obtain four identical dried whole blood microsamples by means of a single capillary sampling. The developed strategy shows significant advantages in terms of blood collection and storage, fast and feasible extraction procedure and sensitive LC-MS/MS analysis, also providing satisfactory validation results (extraction yield >81%, RSD <12.0%, and <6.3% loss in analyte stability after 3 months). The proposed methodology has proven to be sound and reliable for application to the TDM of psychiatric patients treated with antidepressant drugs such as fluoxetine and sertraline. The original capillary volumetric microsampling procedure using hemaPEN has been demonstrated to be suitable for the accurate sampling of capillary whole blood, in order to be successfully exploited in self- and home-sampling procedures in future and to pave the way for precision medicine approaches for the treatment of CNS disorders.
... New sampling devices are now commercially available that can potentially overcome the Hct and volume effects by analysing the entire DBS instead of taking a sub-punch. These new blood collection devices take up and apply a fixed volume of blood onto the filter paper collection device [43][44][45][46][47][48]. Although these new devices are more expensive than the traditional filter paper blood collection devices, their ability to overcome volume and Hct effects would result in improved analytical performance and should be evaluated for the monitoring of patients with IMDs to ensure treatment decisions are made with increased confidence. ...
Monitoring of patients with inherited metabolic disorders (IMDs) using dried blood spot (DBS) specimens has been routinely used since the inception of newborn screening (NBS) for phenylketonuria in the 1960s. The introduction of flow injection analysis tandem mass spectrometry (FIA–MS/MS) in the 1990s facilitated the expansion of NBS for IMDs. This has led to increased identification of patients who require biochemical monitoring. Monitoring of IMD patients using DBS specimens is widely favoured due to the convenience of collecting blood from a finger prick onto filter paper devices in the patient’s home, which can then be mailed directly to the laboratory. Ideally, analytical methodologies with a short analysis time and high sample throughput are required to enable results to be communicated to patients in a timely manner, allowing prompt therapy adjustment. The development of ultra-performance liquid chromatography (UPLC–MS/MS), means that metabolic laboratories now have the capability to routinely analyse DBS specimens with superior specificity and sensitivity. This advancement in analytical technology has led to the development of numerous assays to detect analytes at low concentrations (pmol/L) in DBS specimens that can be used to monitor IMD patients. In this review, we discuss the pre-analytical, analytical and post-analytical variables that may affect the final test result obtained using DBS specimens used for monitoring of patients with an IMD.
... In 2016, BRUSCHETTINI et al. [117] confirmed the importance of limiting the size of blood samples to avoid anaemia due to blood sampling for TDM in preterm infants and highlighted the advantages of DBS over conventional sampling techniques. To overcome the problem of haematocrit, alternative strategies based on new microfluidic sampling procedures or volumetric microsampling devices have been described and proved to be a reliable sampling approach for caffeine [118,119]; however, the drawback is the use of expensive devices for routine TDM analyses or for pharmacokinetic studies. To overcome the problem of blood sample size, invasiveness and cost, in 2017, CHAABANE et al. [120] determined caffeine concentrations in both saliva and serum of preterm infants (mean gestational age 32.2±0.7 weeks), showing a proportional increase in both saliva and serum caffeine concentration to the administered dose, with the saliva caffeine concentrations strongly correlating with those from serum. ...
The incidence of preterm birth is increasing, leading to a growing population with potential long-term pulmonary complications. Apnoea of prematurity (AOP) is one of the major challenges when treating preterm infants; it can lead to respiratory failure and the need for mechanical ventilation. Ventilating preterm infants can be associated with severe negative pulmonary and extrapulmonary outcomes, such as bronchopulmonary dysplasia (BPD), severe neurological impairment and death. Therefore, international guidelines favour non-invasive respiratory support. Strategies to improve the success rate of non-invasive ventilation in preterm infants include pharmacological treatment of AOP. Among the different pharmacological options, caffeine citrate is the current drug of choice. Caffeine is effective in reducing AOP and mechanical ventilation and enhances extubation success; it decreases the risk of BPD; and is associated with improved cognitive outcome at 2 years of age, and pulmonary function up to 11 years of age. The commonly prescribed dose (20 mg·kg ⁻¹ loading dose, 5–10 mg·kg ⁻¹ per day maintenance dose) is considered safe and effective. However, to date there is no commonly agreed standardised protocol on the optimal dosing and timing of caffeine therapy. Furthermore, despite the wide pharmacological safety profile of caffeine, the role of therapeutic drug monitoring in caffeine-treated preterm infants is still debated. This state-of-the-art review summarises the current knowledge of caffeine therapy in preterm infants and highlights some of the unresolved questions of AOP. We speculate that with increased understanding of caffeine and its metabolism, a more refined respiratory management of preterm infants is feasible, leading to an overall improvement in patient outcome.
... Larger DBS samples could also pose issues in the future if the aim is to identify analyte results below an action value instead, for example, low T-receptor excision circles for severe combined immunodeficiency screening and enzyme activities for the screening of galactosemia, biotinidase deficiency and lysosomal storage disorders. Improvement in DBS specimen size and quality could be potentially achieved by the use of blood collection devices that collect defined volumes of liquid blood for sampling or to use approaches to estimate the volume or Hct of a DBS [25][26][27][28]. However, the cost of such strategies to correct for sample volume is at present prohibitive to NBS programs. ...
Aim: Dried blood spots (DBS) are used for the analysis of more than 2000 biomarkers. We assessed a range of analyte concentrations and diameters of DBS. Materials & methods: DBS samples were created by the application of increasing volumes of whole blood prepared by the UK NEQAS Quality Assurance Laboratory. Samples were analyzed in four separate laboratories. Results: Volumes less than 25 μl (8 mm) and more than 75 μl (14 mm) created unsatisfactory analytical biases. Results obtained from peripheral subpunches tended to be higher than those from a central subpunch. Conclusion: DBS diameters formed from nonvolumetric application of blood to filter paper can be used to assess whether measurement bias will be within acceptable limits according to the analyte being quantified. DBS received for newborn screening in the UK with diameters less than 8 mm and those more than 14 mm should be rejected.
... Until recently, the only cellulose-based paper cards have been available for DBS sample collection and just in the last several years a number of new DBS devices like strips, sticks, pens, and tips have been developed [21][22][23][24]. The main disadvantages of DBS are hematocrit effect and a variety of existing commercial DBS matrixes, which have not been yet validated or received regulatory approval for this method of sample collection, transport, and further clinical analysis [25][26][27]. ...
The dried blood spot (DBS) sampling has a lot of advantages in comparison with the “standard” venous blood collecting, such as small collection volume, painless and easy sample collection with minimal training required, stable and transportable at ambient temperatures, etc. The aim of this study was to determine the comparability of four different types of DBS sampling (HemaSpot™-HF Blood Collection Device, Whatman® 903 Protein Saver Snap Apart Card, card ImmunoHealth™, and glass fiber strip ImmunoHealth™) for analysis of the global metabolites profile. All the samples were collected from the same person at the same time and stored at room temperature for four weeks in order to exclude all possible deviations deriving from biological variances and to evaluate sample storage stability. Metabolome profiling by direct injection of a deproteinized capillary blood DBS sample into an electrospray ion source of a hybrid quadrupole time-of-flight mass spectrometer was used. Differences in the metabolomics profile were found between the different DBS collection materials, especially for ImmunoHealth™ card and ImmunoHealth™ glass fiber strip. However, our results indicate that the analytical performance of all tested DBS sampling materials showed consistent results overall detected metabolites and no dramatic changes between them in the metabolic composition during the storage time.
Home‐sampling for therapeutic drug monitoring (TDM) for oral targeted anticancer drugs offers a promising alternative to traditional hospital‐based sampling methods, though it presents challenges. This review aims to summarize the state‐of‐the‐art of home‐sampling methods for TDM and evaluates the analytical and clinical validation challenges. A comprehensive search was conducted across Embase, Medline, and Scopus. Eligible articles described analytical and/or clinical validation of home‐sampling methods for oral targeted anticancer drugs. ASReview was used to process unique references and to identify relevant studies. Of the 39 included articles, 32 detailed on analytical validation experiments, while 27 covered clinical validation experiments. Dried blood spot and volumetric absorptive microsampling were the primary sampling methods. Key challenges were ensuring robust sample collection, sample pretreatment, hematocrit effects, and sample stability, which were generally thoroughly investigated. Clinical validation yielded promising results for most analytes, although external validation remains crucial for confirming reliability. Home‐sampling methods for TDM of oral targeted anticancer drugs show promising results for clinical implementation. Methods for well‐studied drugs may be clinically implemented immediately, while others require further external validation. Future research should address device‐specific challenges and assess patient feasibility to facilitate the routine use of home‐sampling in clinical practice.
Abstract
Background
Dried blood collection devices might be beneficial for diagnosing infectious diseases in areas far from the medical facilities and in lockdown situations. There are several reports on the efficacy of such applications for qualitative tests. Here we demonstrated the feasibility of a novel Ser-Col blood collection device as a standardized approach for qualitative and quantitative detection of infectious markers and several over immunochemical tests.
Methods
In the current study, we included 395 adult participants, 191 men and 204 women, with a median age of 41 years, as well as 75 children with a median age of 3 years. Serological status was determined by testing serum samples for three groups of infection diseases: hepatitis A and C, SARS-CoV-2, and herpes family viruses, as well as for thyroid peroxidase (TPO), prolactin, vitamin B12, and folate. Blood collected on the Ser-Col device (Labonovum) was eluted using an automated system (SCAUT Ser-Col automation, Blok System Supply) and manually. Ser-Col results were compared with serum sampled via standard venipuncture considered as the reference.
Results
High correlation coefficients (r = 0.95–0.99) were observed between serum samples collected with Ser-Col and via standard venipuncture for the following tests: anti-HCV, anti-SARS-CoV-2 IgG, anti-HSV-2 IgG, and anti-CMV IgM. Correlation coefficients between Ser-Col and standard venipuncture serum for anti-HSV-1 IgG, anti-CMV IgG, and anti-EBV tests were relatively low (r = 0.73–0.77). Correlation coefficients for anti-TPO, prolactin, vitamin B12, and folate were also characterized with high values (r = 0.97–0.99).
Conclusions
High accuracy and quantitative correlation were demonstrated between Ser-Col and samples collected by standard venipuncture. Hence, the Ser-Col blood collection device should be considered as a promising alternative for blood collection, storage, and transportation in both adult and pediatric populations.
Self-powered microfluidics presents a revolutionary approach to address the challenges of healthcare in decentralized and point-of-care settings where limited access to resources and infrastructure prevails or rapid clinical decision-making is critical. These microfluidic systems exploit physical and chemical phenomena, such as capillary forces and surface tension, to manipulate tiny volumes of fluids without the need for external power sources, making them cost-effective and highly portable. Recent technological advancements have demonstrated the ability to preprogram complex multistep liquid operations within the microfluidic circuit of these standalone systems, which enabled the integration of sensitive detection and readout principles. This chapter first addresses how the accessibility to in vitro diagnostics can be improved by shifting toward decentralized approaches like remote microsampling and point-of-care testing. Next, the crucial role of self-powered microfluidic technologies to enable this patient-centric healthcare transition is emphasized using various state-of-the-art examples, with a primary focus on applications related to biofluid collection and the detection of either proteins or nucleic acids. This chapter concludes with a summary of the main findings and our vision of the future perspectives in the field of self-powered microfluidic technologies and their use for in vitro diagnostics applications.
A novel concept for highly versatile automated analyses of dried blood spot (DBS) samples by commercial capillary electrophoresis (CE) is presented. Two interchangeable CE cartridges with different fused-silica capillaries were used for the DBS elutions and the DBS eluate analyses, respectively. The application of one CE cartridge with a wide-bore capillary reduced DBS processing times to a minimum (1-2 min per sample) while fitting the other CE cartridge with a narrow-bore capillary served for highly efficient CE analyses. A comprehensive investigation of major variables affecting liquid handling in CE (capillary length, internal diameter, and temperature) was carried out with the aim of optimizing both procedures and enabling their maximum flexibility. The application of two CE cartridges also enabled the employment of CE detectors with different instrumental set-ups and/or principles as was demonstrated by the optical detection of nonsteroidal anti-inflammatory drugs (NSAIDs) and the conductivity detection of amino acids (AAs). The presented methods were optimized for the automated CE analyses of 36 DBS samples formed by a volumetric collection of 5 μL of capillary blood onto Whatman 903 discs and processed by direct in-vial elution using the CE instrument. The precision of liquid transfers for the automated DBS elutions was better than 0.9% and the precision of CE analyses did not exceed 5.1 and 12.3% for the determination of NSAIDs and AAs, respectively. Both methods were linear (R2 ≥ 0.996) over the therapeutic (NSAIDs) and the endogenous (AAs) concentration ranges, had limits of quantification below the typical analyte concentrations in human blood, and achieved sample throughputs of more than 6 DBSs per hour.
Aim: To develop a nondestructive method for the estimation of hematocrit (HCT) in dried blood spots (DBSs). Materials & methods: Standards and controls were created (HCT range: 0.20-0.50 l/l) and DBS scanned using a flatbed scanner. Gray values and pixel areas were analyzed with open-source software to estimate HCT and volume, respectively. HCT obtained in whole blood using hematological analyzer was compared with DBS scanner method (n = 50). Results: Between-run precision was 4.7-10.2% and between-run accuracy was 89.6-102.1%. In the hematological instrument comparison, 96% of the patient sample results were within ±15%. Conclusion: The nondestructive method can be used to exclude patient DBS samples with extreme HCT levels from further analysis and avoid bias on measured concentration.
Analysis of dried urine spots (DUSs) is becoming an emerging technique in clinical, toxicological, and forensic chemistry due to the fully non-invasive collection, facile transportation, and simple storage of DUS samples. Correct DUS collection and elution is of the utmost importance because inadequate DUS sampling/processing may have direct consequences on quantitative DUS analyses and these aspects were, for the first time, comprehensively investigated in this contribution. Various groups of endogenous and exogenous species were selected as model analytes and their concentrations were monitored in DUSs collected on standard cellulose-based sampling cards. Strong chromatographic effects were observed for most analytes having a crucial impact on their distribution within the DUSs during sampling. Concentrations of target analytes were up to 3.75-fold higher in the central DUS sub-punch in comparison to the liquid urine. Consequently, substantially reduced concentrations of these analytes were determined in peripheral DUS sub-punches demonstrating that sub-punching, often applied to dried material spots, is not acceptable for quantitative DUS analyses. Hence, a simple, rapid, and user-friendly procedure was suggested, which employed an in-vial collection of a known urine volume on a pre-punched sampling disc (using a low-cost micropipette designed for patient-centric clinical sampling) and in-vial processing of the whole DUS. Excellent accuracy (0.20%) and precision (0.89%) of liquid transfers were achieved by the micropipette, which was also applied to remote DUS collection by laic and expert users. The resulting DUS eluates were analysed by capillary electrophoresis (CE) for the determination of endogenous urine species. The CE results demonstrated no significant differences between the two user groups, elution efficiencies of 88-100% (in comparison to the liquid urine), and precision better than 5.5%.
Background:
Volumetric absorptive microsampling (VAMS) is an emerging technique that may support multisample collection to enhance therapeutic drug monitoring in solid organ transplantation. This review aimed to assess whether tacrolimus and mycophenolic acid can be reliably assayed using VAMS and to identify knowledge gaps by providing granularity to existing analytical methods and clinical applications.
Methods:
A systematic literature search was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The PubMed, Embase, and Scopus databases were accessed for records from January 2014 to April 2022 to identify scientific reports on the clinical validation of VAMS for monitoring tacrolimus and mycophenolic acid concentrations. Data on the study population, sample sources, analytical methods, and comparison results were compiled.
Results:
Data from 12 studies were collected, including 9 studies pertaining to tacrolimus and 3 studies on the concurrent analysis of tacrolimus and mycophenolic acid. An additional 14 studies that provided information relevant to the secondary objectives (analytical validation and clinical application) were also included. The results of the clinical validation studies generally met the method agreement requirements described by regulatory agencies, but in many cases, it was essential to apply correction factors.
Conclusionss:
Current evidence suggests that the existing analytical methods that use VAMS require additional optimization steps for the analysis of tacrolimus and mycophenolic acid. The recommendations put forth in this review can help guide future studies in achieving the goal of improving the care of transplant recipients by simplifying multisample collection for the dose optimization of these drugs.
Polymeric foams tailor-made of polyvinylpyrrolidone (PVP) and carboxymethylcellulose/oxidized 6-carboxycellulose (CMC07/OC) composite were proposed as suitable sorbents for the collection and analysis of dried blood spots (DBSs). The PVP and CMC07/OC foams were easy to prepare, enabled collection of minute volumes of capillary blood, and blood drying at ambient temperature. The resulting foams were prepared as small porous discs with uniform dimensions (approx. 6 × 3 mm) and were fully soluble in aqueous solutions. The DBSs were formed in standard capillary electrophoresis (CE) vials fitted with the soluble foam discs and enabled the direct in-vial DBS processing and at-line analysis by CE. The DBSs were pretreated with a simple process, which involved a complete dissolution of the foam disc in an acidic solution and a simultaneous hollow fiber liquid-phase microextraction (HF-LPME) in one step. The complete solubility of the foam disc with the DBS served for a quantitative transfer of all blood components into the eluate and a nearly exhaustive HF-LPME of target analytes, whereas the blood matrix and the polymeric foam components were efficiently retained by the organic solvent impregnated in the walls of the HF. The suitability of the PVP and CMC07/OC foams for the collection and the direct analysis of DBSs was demonstrated by the HF-LPME/CE determination of model acidic drugs (warfarin, ibuprofen, naproxen, ketoprofen, and diclofenac) at therapeutically relevant concentrations. Repeatability of the analytical method was better than 8.1% (RSD), extraction recoveries ranged from 70 to 99% (for PVP foam), calibration curves were linear over two orders of magnitude (R2 higher than 0.9991), and limits of detection were less than 44 μg/L (for concentrations in undiluted capillary blood). The soluble polymeric foams exhibited non-significant variations in analyte concentrations for DBSs prepared from blood samples with different hematocrit levels and for aged DBSs (less than 9.2%), moreover, they outperformed standard DBS sampling devices in terms of sample pretreatment time and extraction recovery.
Advances in the technologies to enable patient-centric sampling (PCS) have the potential to improve blood sample collection by enabling clinical trial participants to collect samples via self-collection or with the help of a caregiver in their home. Typically, blood samples to assess pharmacokinetics and pharmacodynamics of a drug during clinical development are collected at a clinical site via venous blood draw. In this position paper by the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ), the potential value PCS can bring to patients, to the clinical datasets generated, and to clinical trial sponsors is discussed, along with considerations for program decision-making, bioanalytical feasibility, operations, and regulatory implications. With an understanding of the value of PCS and considerations when implementing during clinical drug development, we can bring the promise of PCS closer to reality and enable decentralized clinical trials.
Aims
Immunosuppressant and kidney function monitoring are crucial for kidney transplant recipient follow‐up. Microsamples enable remote sampling and minimise patient burden as compared to conventional venous sampling at the clinic. We developed a liquid chromatography–tandem mass spectrometry assay to quantify tacrolimus, mycophenolic acid (MPA), creatinine and iohexol in dried blood spot (DBS), and volumetric absorptive microsample (VAMS) samples.
Methods
The assay was successfully validated analytically for all analytes. Clinical validation was conducted by direct comparison of paired DBS, VAMS and venous reference samples from 25 kidney transplant recipients. Patients received iohexol 5–15 minutes before immunosuppressant intake and were sampled 0, 1, 2 and 3 hours thereafter, enabling tacrolimus and MPA area under the concentration–time curve (AUC) and creatinine‐based and iohexol‐based glomerular filtration rate (GFR) estimation. Method agreement was evaluated using Passing–Bablok regression, Bland–Altman analysis and the percentages of values within 15–30% of the reference (P15–P30) with a P20 acceptance threshold of 80%.
Results
For DBS samples, method agreement was excellent for tacrolimus trough concentrations (n = 25, P15 = 92.0%) and AUCs (n = 25; P20 = 95.8%) and adequate for creatinine‐based GFR trend monitoring (n = 25; P20 = 80%). DBS‐based MPA AUC assessment showed suboptimal agreement (n = 16; P20 = 68.8%), but was considered acceptable given its P30 of 100%. The assay performed inadequately for DBS‐based iohexol GFR determination (n = 24; P20 = 75%). The VAMS technique generally showed inferior performance, but can be considered for certain situations.
Conclusion
The assay was successfully validated for tacrolimus, MPA and creatinine quantification in DBS samples, enabling simultaneous remote kidney function trend monitoring and immunosuppressant therapeutic drug monitoring in kidney transplant recipients.
Résumé
L’utilisation des spots de sang séché en biologie remonte aux travaux de Guthrie appliqués initialement au dépistage néonatal de la phénylcétonurie, puis étendus depuis au dépistage d’une dizaine de maladies héréditaires. Initialement qualitative ou semi-quantitative, la méthode des spots de sang séché (DBS – Dried Blood Spot) a été appliquée à la quantification de nombreux analytes, avec une difficulté majeure due au manque de précision du volume de sang prélevé. Parmi les facteurs influençant ce volume, l’hématocrite est sans nul doute le plus limitant. Deux nouveaux systèmes dits volumétriques, HémaPENTM et MitraTM ont permis de s’affranchir de la valeur de l’hématocrite. Ils permettent de prélever de façon précise des volumes compris entre 2,5 et 30 µL. Comme les DBS, ces nouveaux systèmes présentent de nombreux avantages comme la facilité de transport et de stockage. Cependant, les faibles volumes de sang prélevés, s’ils sont particulièrement adaptés aux études en pédiatrie ou à l’expérimentation animale, nécessitent le développement de méthodes analytiques sensibles. Ces méthodes sont en général des méthodes chromatographiques couplées à de la spectrométrie de masse validées avec une phase de préparation d’échantillon plus longue que pour des prélèvements classiques. Malgré leurs couts élevés, les systèmes volumétriques ont permis de développer de nombreuses applications quantitatives dans de multiples domaines de la biologie, comme la pharmacocinétique, le suivi thérapeutique, la toxicologie clinique et médico-légale.
Dried blood spot (DBS) sampling by finger-pricking has recently gained a lot of interest as an alternative sample collection method. The reduced invasiveness, requirement of lower sample volumes and suitability for long-term storage at room temperature make DBS ideal for use in home settings or low-resource environments. However, traditional protocols often suffer from biased analysis data due to variable and not exactly known blood volumes present in the samples. In this work, a novel device has been developed to split-off precisely metered volumes from a blood drop and load them on pre-cut filter paper. Hereto, hydrophobic burst valves (HBV) were developed to temporarily retain a fluid flow, configurable to burst at pressures within a range of 175-600 Pa. By combining HBVs with different burst pressures, a volume metering system was developed to allow parallel metering of multiple pre-defined sample volumes. The system was shown to be accurate and consistent for blood volumes between 5-15 μL and for hematocrit levels spanning the range of 25-70%. Finally, a point-of-care DBS sampling device was developed combining the self-powered microfluidic SIMPLE technology. To evaluate the system's practical applicability, a validation study in the context of therapeutic drug monitoring of biologicals was performed using adalimumab-spiked blood samples. Microfluidic DBS samples showed good performance compared to the traditional DBS method with improved recovery rates (86% over 62%). This innovative metering system, allowing for parallelization and integration with complex liquid manipulations, will greatly impact the field of robust sampling, sample preparation, storage and analysis at the point-of-care.
The performance of state-of-the-art high-resolution continuum source graphite furnace atomic absorption spectrometry (HR CS GFAAS) instrumentation and four novel devices to produce dried blood spots of perfectly defined volumes is evaluated with the aim of developing a simple, direct method for the determination of Cu in blood samples. In all cases, it is feasible to obtain accurate quantitative information using any of the four devices tested (Mitra, HemaXis DB10, Capitainer qDBS and HemaPEN) via simple external calibration with aqueous standards. One of the main differences in the performance of such devices is related to the blanks obtained, such that HemaXis DB10 and HemaPEN are preferred when abnormally low Cu levels (500 μg L-1 or lower), associated with some diseases, need to be determined. The results prove that accurate values with RSD values below 10% can be achieved for these devices even for such Cu levels, while for Capitainer qDBS and, to a higher extent, for Mitra, blank variations will ultimately increase the uncertainty. It is important to stress that analysis of four real samples using both venipuncture and all the DBS specimens showed a very good agreement. Thus, the approach proposed could be readily applied, such that patients with disorders requiring Cu control can prepare their own samples and submit them via postal mail to labs for HR CS GFAAS direct and fast analysis.
The outbreak of the new coronavirus disease changed the world upside down. Every day, millions of people were subjected to diagnostic testing for Covid‐19, all over the world. Molecular tests helped in the diagnosis of current infection by detecting the presence of viral genome whereas serological tests helped in detecting the presence of antibody in blood as well as contributed to vaccine development. This testing helped in understanding the immunogenicity, community prevalence, geographical spread, conditions post‐infection. However, with the contagious nature of the virus, biological specimen sampling involved the risk of transmission and spread of infection. Clinic or pathology visit was the most concerning part. Trained personnel and resources was another barrier. In this scenario, microsampling played an important role due to its most important advantage of remote, contactless, small volume, self‐sampling. Minimum requirements for sample storage and ease of shipment added value in this situation. The highly sensitive instruments and validated assay formats assured the accuracy of results and stability of samples. Microsampling techniques are contributing effectively to the Covid‐19 pandemic by reducing the demand for clinical staff in population‐level testing. The validated and established applications supported the use of microsampling in diagnosis, therapeutic drug monitoring, development of treatment or vaccines, clinical trials for Covid‐19.
Au cours des cinquante dernières années, le dépistage néonatal est devenu incontournable dans les programmes de santé publique de très nombreux pays. Durant toutes ces années, le nombre de maladies susceptibles d’être dépistées à la naissance n’a cessé de croître grâce aux possibilités offertes par les progrès techniques de la biologie clinique. Le test de Guthrie a permis la miniaturisation du prélèvement de sang, permettant ainsi le dépistage biologique dans la population des nouveau-nés. Par la suite, la fluorimétrie, l’immunoanalyse et, plus récemment, la spectrométrie de masse en tandem ont rendu possible le dépistage de nombreuses maladies qu’il est possible de traiter. Les nouvelles possibilités de séquençage du génome et d’intelligence artificielle vont probablement ouvrir une nouvelle ère, malgré les nombreuses questions éthiques qui se poseront. Cette revue propose de dresser le panorama des techniques biologiques utilisées actuellement pour le dépistage néonatal et de mettre en perspective la place de nouvelles évolutions techniques.
An effective signal amplification strategy is essential to enhance the analytical performance of microfluidic paper-based analytical devices (μPADs) for tracing biomarkers. Here, a simple but efficient approach with superior electrocatalytic performance of Pd@hollow Zn/Co core-shell ZIF67/ZIF8 nanoparticles for regulating the efficacious signal amplification process was utilized to realize the detection of prostate-specific antigen (PSA). By rationally designing the core-shell structure of ZIF67/ZIF8 with hollow characteristics on the nanoscale and introducing the noble metal element Pd into the cavity, the diffusion limitation and porous confinement reduction of the obtained nanomaterials with uniform morphology and satisfactory chemical stability could be realized, which endowed it with better catalytic performance than solid metal-organic frameworks (MOFs) and ensured effective signal amplification of H2O2 reduction for achieving enhanced electrochemical signals. Moreover, with the assistance of signal probes, the remaining H2O2 could flow to the color area to catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine to form a colored product by changing the spatial configuration of the μPAD, thus realizing the visual detection of PSA. On the basis of this novel analytical device, dual-mode ultrasensitive detection of PSA could be achieved with a lower limit of detection of 0.78 pg/mL (S/N = 3) and a wider linear range from 5 pg/mL to 50 ng/mL. This work provided the opportunity of introducing the noble metal element Pd into the cavity of the MOF hollow structure to improve its electrocatalytic efficiency and construct a high-performance μPAD for clinical detection of other biomarkers.
Purpose:
The use of microsampling for therapeutic drug monitoring (TDM) is increasingly feasible as sensitive methods have become more accessible. There exists an increasing interest in the use of microsampling, and new microsampling devices and techniques can potentially improve patient convenience and care, amongst other features. This review provides an update on currently validated methods for measuring drugs pertinent to TDM, including data from clinical samples.
Methods:
A literature record search was undertaken, including PubMed and Google Scholar. Reports that included the use of microsampling to measure concentrations of drugs associated with TDM were reviewed and included if data from patient samples were reported. The studies are described in brief, including sample preparation and analyte stability, with the most pertinent findings reported.
Results:
Sensitive analyses and innovative designs and materials have resulted in an increasing number of reported evaluations and validations for measuring drugs using microsamples. Novel designs largely overcome common problems associated with traditional dried blood spot sampling. Although examples of patient self-sampling are rare at present, studies that evaluated feedback found it to be largely positive, revealing the feasibility of microsampling for TDM.
Conclusions:
Microsampling is suited to the TDM of numerous drugs in diverse situations, and will play an increasingly important role. The issues with traditional dried blood spot samples have been largely overcome by employing novel methods to obtain volumetric samples.
Background: The hemaPEN is a liquid microsampling device for the reproducible collection and storage of blood samples as dried blood spots, for subsequent quantitative analysis. Materials & methods: We examined the device’s ability to collect accurate and precise blood volumes, at different hematocrit levels, via in vitro studies using acetaminophen in human blood. We also investigated the impact of different user training approaches on device performance. Results: The hemaPEN demonstrated acceptable volumetric accuracy and precision, regardless of the training medium used. Issues with apparent hematocrit-dependent bias were found to be associated with the extraction process, rather than the volumetric performance of the device. Conclusion: The hemaPEN is capable of readily producing high quality blood microsamples for reproducible and accurate quantitative bioanalysis.
Background:
Dried blood spots (DBS) has gained recent popularity as a sampling method for therapeutic drug monitoring (TDM). For patients, DBS sampling has several advantages over venous blood sampling. However, technical issues primarily influenced by hematocrit levels, interfere with the implementation of this method in daily clinical practice. The results of concentration measurements of drugs that are influenced by hematocrit should be corrected for hematocrit levels. Here, we developed a fast, nondestructive, near-infrared (NIR)-based method for measuring the hematocrit in DBSs.
Method:
Using a partial least squares (PLS) algorithm, an NIR-based quantification method was developed for measuring hematocrit levels of 0.19-0.49 L/L. Residual venous blood of 522 patients was used to build this PLS model. The validity of the method was evaluated using 40 patient samples. DBS were created by adding a small amount (50 µL) of blood on a Whatman filter paper and drying for 24 hours in a desiccator cabinet. The robustness was evaluated by measuring 24 additional samples with a high hemolysis, icterus, and lipemia index (HIL index). The hematocrit values obtained using a Sysmex XN hemocytometry analyzer were used as reference.
Results:
The difference between hematocrit measurements obtained with NIR spectroscopy and a hemocytometry analyzer was <15% for the 40 samples. The accuracy (≤9%) and precision (≤7%) for all the quality control (QC) samples were within the acceptance criteria of <15%. The intra-assay and inter-assay coefficient of variability (CV) was ≤3% and ≤6%, respectively, for the different QC levels. There were no deviations in the measurements for the samples with high HIL indices. The stability of hematocrit in DBS was up to 14 days for all levels.
Conclusion:
We developed and validated a hematocrit model using NIR spectroscopy. This nondestructive, accurate, and reproducible method has a short analysis time (51 s), and can be used to analyze DBS samples stored for up to 2 weeks in a desiccator cabinet.
In analytical toxicology, no matter how complex the equipment and careful the analysis, the results may be rendered worthless if sample collection, transport, and storage have not been performed with the analysis in mind. Biological samples may contain infective agents and must be handled with care, especially if originating from substance misusers, and must always be treated as if they are infective. Clinical samples can be divided into blood and related fluids, body fluids other than blood, excretory fluids/residues, and other clinical specimens. This chapter presents a list of some clinical sample types and provides guidelines for sample collection for analytical toxicology. It discusses the advantages and disadvantages of different sample types in analytical toxicology. The chapter provides information on the sample transport, storage, and disposal, as well as common interferences encountered in the storage of samples.
Use of dried matrix spots, such as dried blood spots -DBS- and dried urine spots -DUS-, is becoming more popular in clinical analyses, beyond their traditional use for sampling newborns. Currently, there are new types of microsampling devices that have been designed to collect a low and known volume of biological fluids, regardless of the hematocrit level, thus making it feasible to develop fully quantitative methods for their analysis. In this study, three of the most promising microsampling devices (Capitainer B, Hemaxis DB 10 and Mitra) were evaluated aiming at the direct determination of Hg using a dedicated “analyzer” based on atomic absorption spectrometry. Whole blood and urine reference materials were used to evaluate the methods developed. This Hg analyzer possesses a gold trap to enhance sensitivity and minimize matrix effects, enabling direct analysis of the solid DBS and DUS samples using calibration with Hg aqueous solutions, with an instrumental limit of detection (LOD) and quantification (LOQ) of 3.4 and 11 pg, respectively. LOQs when using the three microsampling devices were similar, ranging between 2.5 and 3.2 µg L–1. In case of need, it is demonstrated that the simultaneous analysis of four Mitra derived DBS at the same time in a quartz boat enables decreasing the LOD to 0.32 µg L–1.
Background:
An LC-MS/MS method has been developed for the simultaneous quantification of P-glycoprotein (P-gp) and cytochrome P450 (CYP) probe substrates and their Phase I metabolites in DBS and plasma. P-gp (fexofenadine) and CYP-specific substrates (caffeine for CYP1A2, bupropion for CYP2B6, flurbiprofen for CYP2C9, omeprazole for CYP2C19, dextromethorphan for CYP2D6 and midazolam for CYP3A4) and their metabolites were extracted from DBS (10 µl) using methanol. Analytes were separated on a reversed-phase LC column followed by SRM detection within a 6 min run time.
Results:
The method was fully validated over the expected clinical concentration range for all substances tested, in both DBS and plasma. The method has been successfully applied to a PK study where healthy male volunteers received a low dose cocktail of the here described P-gp and CYP probes. Good correlation was observed between capillary DBS and venous plasma drug concentrations.
Conclusion:
Due to its low-invasiveness, simple sample collection and minimal sample preparation, DBS represents a suitable method to simultaneously monitor in vivo activities of P-gp and CYP.
Dried blood spot (DBS) sampling for quantitative determination of drugs in blood has entered the bioanalytical arena at a fast pace during the last decade, primarily owing to progress in analytical instrumentation. Despite the many advantages associated with this new sampling strategy, several issues remain, of which the hematocrit issue is undoubtedly the most widely discussed challenge, since strongly deviating hematocrit values may significantly impact DBS-based quantitation. In this review, an overview is given of the different aspects of the 'hematocrit problem' in quantitative DBS analysis. The different strategies that try to cope with this problem are discussed, along with their potential and limitations. Implementation of some of these strategies in practice may help to overcome this important hurdle in DBS assays, further allowing DBS to become an established part of routine quantitative bioanalysis.
Background:
The European Bioanalysis Forum dried blood spots (DBS)/microsampling consortium is reporting back from the experiments they performed on further documenting the potential hurdles of the DBS technology. This paper is focused on the impact of hematocrit changes on DBS analyses.
Results:
The hematocrit can have an effect on the size of the blood spot, on spot homogeneity and on extraction recovery in a compound-dependent manner. The extraction recovery can change upon aging in an hematocrit-dependent way. Different card materials can give different outcomes.
Conclusions:
The results from the conducted experiments show that the issues of DBS in regulated bioanalysis are real and that the technology will need improvements to be ready for use as a general tool for regulated bioanalysis.
A sensitive, specific and efficient high-performance liquid chromatography-tandem mass spectrometry assay for the simultaneous determination of vincristine and actinomycin-D in human dried blood spots is presented. Dried blood spots were punched out of a collection paper with a 0.25-in.-diameter punch. The analytes were extracted from the punched-out disc using sonication during 15 min in a mixture of acetonitrile-methanol-water (1:1:1, v/v/v) containing the internal standard vinorelbine. Twenty-microlitre volumes were injected onto the HPLC system. Separation was achieved on a 50 x 2.1 mm ID Xbridge C(18) column using elution with 1 mM ammonium acetate-acetonitrile (70:30, v/v) adjusted to pH 10.5 with ammonia and run in a gradient with methanol at a flow rate of 0.4 mL/min. HPLC run time was 6 min. The assay quantifies vincristine from 1 to 100 ng/mL and actinomycin-D from 2 to 250 ng/mL using a blood sample obtained by a simple finger prick. Validation results demonstrate that vincristine and actinomycin-D can be accurately and precisely quantified in human dried blood spots with the presented method. The assay can now be used to support clinical pharmacologic studies with vincristine and actinomycin-D.
Dried blood spots (DBS) typically consist in the deposition of small volumes of capillary blood onto dedicated paper cards. Comparatively to whole blood or plasma samples, their benefits rely in the fact that sample collection is easier and that logistic aspects related to sample storage and shipment can be relatively limited, respectively, without the need of a refrigerator or dry ice. Originally, this approach has been developed in the sixties to support the analysis of phenylalanine for the detection of phenylketonuria in newborns using bacterial inhibition test. In the nineties tandem mass spectrometry was established as the detection technique for phenylalanine and tyrosine. DBS became rapidly recognized for their clinical value: they were widely implemented in pediatric settings with mass spectrometric detection, and were closely associated to the debut of newborn screening (NBS) programs, as a part of public health policies. Since then, sample collection on paper cards has been explored with various analytical techniques in other areas more or less successfully regarding large-scale applications. Moreover, in the last 5 years a regain of interest for DBS was observed and originated from the bioanalytical community to support drug development (e.g., PK studies) or therapeutic drug monitoring mainly. Those recent applications were essentially driven by improved sensitivity of triple quadrupole mass spectrometers. This review presents an overall view of all instrumental and methodological developments for DBS analysis with mass spectrometric detection, with and without separation techniques. A general introduction to DBS will describe their advantages and historical aspects of their emergence. A second section will focus on blood collection, with a strong emphasis on specific parameters that can impact quantitative analysis, including chromatographic effects, hematocrit effects, blood effects, and analyte stability. A third part of the review is dedicated to sample preparation and will consider off-line and on-line extractions; in particular, instrumental designs that have been developed so far for DBS extraction will be detailed. Flow injection analysis and applications will be discussed in section IV. The application of surface analysis mass spectrometry (DESI, paper spray, DART, APTDCI, MALDI, LDTD-APCI, and ICP) to DBS is described in section V, while applications based on separation techniques (e.g., liquid or gas chromatography) are presented in section VI. To conclude this review, the current status of DBS analysis is summarized, and future perspectives are provided. © 2014 Wiley Periodicals, Inc. Mass Spec Rev 9999:1–78, 2014
Volumetric Absorbtive Micro Sampling is a novel approach to obtaining a dried blood sample for quantitative bioanalysis that overcomes the area bias and sample homogeneity issues associated with conventional DBS when a sub-punch is taken from the sample. The VAMS sampler absorbs a fixed volume of blood (~10 µL) in 2 to 4 seconds with less than 5% volume variation across the hematocrit range 20 to 70% with low tip to tip variability. There is no evidence of selective absorption by the tip of the plasma component over whole blood. Recommendations for best practice when collecting samples were developed based upon the results of tests examining a number of potential abuse scenarios.
Bioanalysis Zone carried out a survey to evaluate the use of and attitudes to DBS analysis among our readership in the bioanalytical community. DBS analysis has generated a huge amount of interest in recent years. We wanted to take a snapshot of the field and determine whether a consensus is emerging on the future of DBS. We asked you for your honest opinions and you delivered! You can view the full results at www.bioanalysis-zone.com. We have a selection of short opinion articles below, giving a range of viewpoints on the current and future use of DBS, alongside some of the survey results.
Background:
To ensure that PK data generated from DBS samples are of the highest quality, it is important that the paper substrate is uniform and does not unduly contribute to variability. This study investigated any within and between lot variations for four cellulose paper types: Whatman™ FTA(®) DMPK-A, -B and -C, and 903(®) (GE Healthcare, Buckinghamshire, UK). The substrates were tested to demonstrate manufacturing reproducibility (thickness, weight, chemical coating concentration) and its effect on the size of the DBS produced, and the quantitative data derived from the bioanalysis of human DBS samples containing six compounds of varying physicochemical properties.
Results & discussion:
Within and between lot variations in paper thickness, mass and chemical coating concentration were within acceptable manufacturing limits. No variation in the spot size or bioanalytical data was observed.
Conclusion:
Bioanalytical results obtained for DBS samples containing a number of analytes spanning a range of chemical space are not affected by the lot used or by the location within a lot.
Dried blood spots have become a popular method in a variety of micro blood-sampling techniques in the life sciences sector, consequently competing with the field of conventional, invasive blood sampling by venepuncture. Dried blood spots are widely applied in numerous bioanalytical assays and have gained a significant role in the screening of inherited metabolic diseases, in PK and PD modeling; in the treatment and diagnosis of infectious diseases; and in therapeutic drug monitoring. Recent technological developments such as automation, online extraction, mass spectrometric direct analysis and also conventional dried blood spot bioanalysis, as well as future developments in dried blood spot bioanalysis are highlighted and presented in this article.
Background:
The hematocrit effect is a hurdle for successful introduction of the dried blood spot (DBS) in a regulated environment. Recently, attempts were taken to overcome the hematocrit effect by whole-cut DBS analysis. This paper presents the next-generation whole-cut DBS; dried matrix on paper disks (DMPD).
Results:
DMPD eliminated the hematocrit effect and demonstrated better accuracy and precision than regular DBS with partial punching. Observed accuracy and precision were 6.0 and 2.3% for DMPD, respectively, and -10.4 and 17.1%, for DBS, respectively.
Conclusion:
The DMPD technique performed better than regular DBS by eliminating the hematocrit effect related blood volume bias. Although this effect was not observed with DMPD, a systematic error of 6.0% was detected and further technical development of DMPD could improve the performance.
Tyrosinemia type 1 is caused by deficiency of fumarylacetoacetate hydrolase. The enzymatic defect impairs the conversion of fumarylacetoacetate to fumarate, causing accumulation of succinylacetone which induces severe liver and kidney dysfunction along with mutagenic changes and hepatocellular carcinoma. Treatment is based on nitisinone (NTBC), an enzymatic inhibitor which suppresses succinylacetone production. NTBC, which has dramatically changed the disease course improving liver and kidney functions and reducing risk of liver cancer, causes a side effect of the increase of tyrosine levels. Treatment is therefore based on the combination of NTBC with a protein-restricted diet to prevent the potential toxicity of excessive tyrosine accumulation. Long-term therapy requires a careful monitoring in blood of NTBC levels along with other disease biomarkers, which include succinylacetone, and a selected panel of circulating aminoacids. We have developed a straightforward and fast MS/MS method for the simultaneous determination of NTBC, succinylacetone, tyrosine, phenylalanine, and methionine on a dried blood spot requiring a 2 min run. A single assay suitable for quantitative evaluation of all biochemical markers is of great advance over conventional methods, especially in pediatric patients, since it reduces laboratory costs and blood sampling, is less invasive and particularly suitable for pediatric patients, and allows easier storage and shipping.
Dried blood spot (DBS) technology was evaluated for the quantitative determination of guanfacine in human blood in clinical studies. A very sensitive DBS assay has been developed using HPLC coupled with an AB Sciex 5500 QTRAP® (Applied Biosystems/MDS Sciex, ON, Canada) MS system (LC-MS/MS) with a linear calibration range of 0.05 to 25 ng/ml. High-resolution MS using an Exactive Orbitrap® (ThermoFisher, LLC., CA, USA) was compared with the QTRAP using extracted exact mass ion current profiles for guanfacine and its stable-isotope-incorporated internal standard. The sample preparation employed liquid-liquid extraction with methyl t-butyl ether of 5 mm punched DBS card disks, followed by reversed-phase HPLC separation coupled with either MS/MS or high-resolution MS. Routine experiments were performed to establish the robustness of the DBS assay, including precision, accuracy, linearity, selectivity, sensitivity and long-term stability of up to 76 days. In addition, several factors that potentially affect quantitation were investigated, including blood volume for DBS spotting, punch size and punch location.
A sensitive research assay with a LLOQ of 0.05 ng/ml was developed and subjected to several components of a method validation common to a regulated bioanalysis procedure employing DBS. This method development and partial validation study determined that spot volume, punch size or punch location do not affect assay accuracy and precision. The DBS approach was successfully applied to a clinical study (a Phase I, randomized, double-blind, placebo-controlled, crossover study to assess the effect of varying multiple oral doses of guanfacine on the pharmacokinetic, pharmacodynamic, safety, and tolerability profiles in healthy adult subjects). The pharmacokinetic profiles for 12 volunteers generated from the DBS assay and from a previously validated plasma assay were compared and were found to be comparable. DBS incurred samples collected from finger prick blood and directly applied to the DBS cards were also analyzed for comparison.
From a bioanalytical perspective, DBS sample collection and analysis is a potentially viable alternative for guanfacine determination in clinical studies, utilizing approximately 100 µl of blood per subject profile compared with a few millilitres of blood drawn for conventional plasma bioanalysis.
Dried blood spots are becoming a popular alternative to plasma for many different applications. This has been driven by animal ethics but also by ease of use and cost savings. Recent regulatory guidance now has a requirement for incurred sample reanalysis. This article details three examples of incurred sample reanalysis using dried blood spot samples.
A novel approach has been developed for the quantitative determination of circulating drug concentrations in clinical studies using dried plasma spots (DPS) on paper substrates, rather than conventional plasma samples. A quantitative bioanalytical high-pressure liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay has been validated using paroxetine as a tool compound (range 0.2-200 ng/mL human plasma). The assay employed simple solvent extraction of a punched disk taken from the DPS sample, followed by reversed phase HPLC separation, combined with multiple reaction monitoring mass spectrometric detection. In addition to performing routine experiments to establish the validity of the assay to internationally accepted criteria (precision, accuracy, linearity, sensitivity, selectivity), experiments are included to assess the effect of the volume of plasma spotted and the use of an indicating paper. The validated DPS approach was successfully applied to a clinical study utilizing pooled samples and a direct comparison of DPS and plasma was made (single oral dose of 37.5 mg of paroxetine).
There are little published data on either the comparison of liquid blood and dried blood spots (DBS) analyses or the ability to generate comparable DBS data at different analytical laboratories. We assess the comparative results of samples stored as liquid blood and DBS. We also determine the transferability of DBS samples by comparing the analysis at two laboratories.
Bioanalytical methods for the analysis of pioglitazone in DBS and liquid blood samples were validated to US FDA guidelines. Pharmacokinetic data generated from DBS and liquid blood samples demonstrated area under the time-concentration profile (0-24 h) values within 3% of each other and maximum plasma concentration values within 7% of each other. Comparing DBS sample results at different laboratories showed more than 99% of results agreeing within 20%.
The results indicate that comparable concentration results are obtained from DBS and whole blood samples within the same laboratory, indicating that changing between the two matrices is viable. The comparable results of DBS samples analyzed at two laboratories using different analytical methodologies demonstrate that the technique is robust and transferable.
As hematocrit levels are known to vary between individuals and with disease state, its effect on the physical characteristics of dried blood spot (DBS) samples and on the accurate quantification of analytes within these samples is examined.
The area of DBS samples decreases with increasing hematocrit levels in a linear manner on the three cellulose paper substrates tested. Furthermore, a bias was observed in the concentrations of two analytes determined in DBS samples at different hematocrits, which in some cases exceeded acceptable values, particularly for hematocrits outside normal values.
If it is expected that the hematocrit of study samples will vary from values considered normal, then its effect on the quantitative determination of an analyte in DBS samples should be investigated as part of the method development and validation. If an unacceptable effect is observed, then this will need to be addressed, by modification of the analytical method, or the inclusion of quality control samples at different hematocrit levels to show control of the assay.
Rufinamide (RUF) is a new antiepileptic drug with efficacy in several types of seizures. The aim of this study was to evaluate the use of dried blood spot (DBS) specimens to determinate RUF levels during treatment. Therapeutic drug monitoring of RUF could be useful in routine clinical practice. Advantages of DBS include short collection time, low invasiveness, ease and low cost of sample collection, transport and storage. The analysis was performed in selected reaction monitoring (SRM) mode. The calibration curve in matrix was linear in the concentration range of 0.008-0.8 mg/L (0.48-47.60 mg/L in DBS) of rufinamide with correlation coefficient value of 0.996. In the concentration range of 0.48-47.6 mg/L, the coefficients of variation in DBS were in the range 1.58-4.67% and the accuracy ranged from 89.73% to 107.32%. The sensitivity and specificity of tandem mass spectrometry allow now high throughput rufinamide analysis. This new assay has favourable characteristics being highly precise and accurate. The published HPLC-UV methods also proved to be precise and accurate, but required not less than 0.2-0.5 mL of plasma and are therefore unsuitable for sample collection in neonates in whom obtaining larger blood samples is not convenient or possible.
A new method is described for rapid and economical screening of large numbers of hospital nursery infants for elevation in blood phenylalanine associated with phenylketonuria. Results are presented for 682 infants, 96% of whom were 4 days of age. None of the blood phenylalanine values were found to be as high as 4 mg/100 ml, and only 8% were above 2 mg/100 ml. These values appear to be in agreement with values obtained by other methods, and indicate that a very low rate of "false-positives" will be encountered during screening of the 10,000 or more infants that may be necessary to detect a case of phenylketonuria. It is recommended that any result of 6 mg/100 ml or above be considered positive, and require confirmation by phenylalanine determination of a second blood specimen.
- G Marca
- S Malvagia
- L Filippi
- M Innocenti
- A Rosati
- M Falchi
- S Pellacani
- G Moneti
- R Guerrini
Marca, G.; Malvagia, S.; Filippi, L.; Innocenti, M.; Rosati, A.;
Falchi, M.; Pellacani, S.; Moneti, G.; Guerrini, R. J. Pharm. Biomed.
Anal. 2011, 54, 192−197.
(6) la Marca, G.; Malvagia, S.; Materazzi, S.; Della Bona, M. L.;
Boenzi, S.; Martinelli, D.; Dionisi-Vici, C. Anal. Chem. 2012, 84,
1184−1188.
- C W N Damen
- H Rosing
- J H M Schellens
- J H Beijnen
- S Ahmad
- M Bioanalysis Busz
- P E Turpin
- J E C Burnett
- L Goodwin
- A Foster
- De
- P M M Kesel
- N Sadones
- S Capiau
- W Lambert
- R De Vries
- M Barfield
- N Van De Merbel
- B Schmid
- C Siethoff
- J Ortiz
- E Verheij
Damen, C. W. N.; Rosing, H.; Schellens, J. H. M.; Beijnen, J. H.
Anal. Bioanal. Chem. 2009, 394, 1171−1182.
(10) Barfield, M.; Ahmad, S.; Busz, M. Bioanalysis 2011, 3, 1025−
1030.
(11) Turpin, P. E.; Burnett, J. E. C.; Goodwin, L.; Foster, A.; Barfield,
M. Bioanalysis 2010, 2, 1489−1499.
(12) De Kesel, P. M. M.; Sadones, N.; Capiau, S.; Lambert, W. E.;
Stove, C. P. Bioanalysis 2013, 5, 2023−2041.
(13) de Vries, R.; Barfield, M.; van de Merbel, N.; Schmid, B.;
Siethoff, C.; Ortiz, J.; Verheij, E.; van Baar, B.; Cobb, Z.; White, S.;
Timmerman, P. Bioanalysis 2013, 5, 2147−2160.
(14) Denniff, P.; Spooner, N. Bioanalysis 2010, 2, 1385−1395.
(15) De Kesel, P. M. M.; Capiau, S.; Lambert, W. E.; Stove, C. P.
Bioanalysis 2014, 6, 1871−1874.
(16) Fan, L.; Lee, J. A. Bioanalysis 2012, 4, 345−347.
(17) Denniff, P.; Spooner, N. Anal. Chem. 2014, 86, 8489−8495.
- Y Y Li
- J Henion
- R Abbott
- P Wang
Li, Y. Y.; Henion, J.; Abbott, R.; Wang, P. Bioanalysis 2011, 3,
2501−2514.
(8) Barfield, M.; Wheller, R. Anal. Chem. 2011, 83, 118−124.
- J Luckwell
- P Denniff
- S Capper
- P Michael
- N Spooner
- P Mallender
- B Johnson
- S Clegg
- M Green
- S Ahmad
- L Woodford
- R J Meesters
- G P Hooff
Luckwell, J.; Denniff, P.; Capper, S.; Michael, P.; Spooner, N.;
Mallender, P.; Johnson, B.; Clegg, S.; Green, M.; Ahmad, S.;
Woodford, L. Bioanalysis 2013, 5, 2613−2630.
(3) Meesters, R. J.; Hooff, G. P. Bioanalysis 2013, 5, 2187−2208.
(4) Guthrie, R.; Susi, A. Pediatrics 1963, 32, 338−343.
- S Ahmad
- M Busz
- P E Bioanalysis Turpin
- J E C Burnett
- L Goodwin
- W E Lambert
Anal. Bioanal. Chem. 2009, 394, 1171−1182.
(10) Barfield, M.; Ahmad, S.; Busz, M. Bioanalysis 2011, 3, 1025−
1030.
(11) Turpin, P. E.; Burnett, J. E. C.; Goodwin, L.; Foster, A.; Barfield,
M. Bioanalysis 2010, 2, 1489−1499.
(12) De Kesel, P. M. M.; Sadones, N.; Capiau, S.; Lambert, W. E.;
- C Siethoff
- J Ortiz
- E Verheij
- B Van Baar
- Z Cobb
- S White
- P Timmerman
- P Denniff
- N Spooner
- P M M Bioanalysis De Kesel
- S Capiau
- W E Lambert
- C P Stove
- L Fan
- J A Lee
- P Denniff
- N Spooner
- G P Hooff
Siethoff, C.; Ortiz, J.; Verheij, E.; van Baar, B.; Cobb, Z.; White, S.;
Timmerman, P. Bioanalysis 2013, 5, 2147−2160.
(14) Denniff, P.; Spooner, N. Bioanalysis 2010, 2, 1385−1395.
(15) De Kesel, P. M. M.; Capiau, S.; Lambert, W. E.; Stove, C. P.
Bioanalysis 2014, 6, 1871−1874.
(16) Fan, L.; Lee, J. A. Bioanalysis 2012, 4, 345−347.
(17) Denniff, P.; Spooner, N. Anal. Chem. 2014, 86, 8489−8495.
(18) Wagner, M.; Tonoli, D.; Varesio, E.; Hopfgartner, G. Mass. Spec.
Rev. 2014, DOI: 10.1002/mas.21441.
(19) Bosilkovska, M.; Deglon, J.; Samer, C.; Walder, B.; Desmeules,
J.; Staub, C.; Daali, Y. Bioanalysis 2014, 6, 151−164.
(20) Meesters, R. J. W.; Zhang, J.; van Huizen, N. A.; Hooff, G. P.;
- P Denniff
- C Holliman
- L Svensson
- N Weng
- S Patel
- Bioanalysis
REFERENCES
(1) Denniff, P.; Holliman, C.; Svensson, L.; Weng, N.; Patel, S.
Bioanalysis 2014, 6, 287−291.
(2) Luckwell, J.; Denniff, P.; Capper, S.; Michael, P.; Spooner, N.;
Bioanalysis 2013, 5, 2023−2041. (13) de Vries
- C P Stove
Stove, C. P. Bioanalysis 2013, 5, 2023−2041.
(13) de Vries, R.; Barfield, M.; van de Merbel, N.; Schmid, B.;
- S Boenzi
- D Martinelli
- C Dionisi-Vici
- Y Y Li
- J Henion
- R Abbott
- P Wang
Boenzi, S.; Martinelli, D.; Dionisi-Vici, C. Anal. Chem. 2012, 84,
1184−1188.
(7) Li, Y. Y.; Henion, J.; Abbott, R.; Wang, P. Bioanalysis 2011, 3,
2501−2514.
(8) Barfield, M.; Wheller, R. Anal. Chem. 2011, 83, 118−124.
(9) Damen, C. W. N.; Rosing, H.; Schellens, J. H. M.; Beijnen, J. H.
- L Woodford
- R J Bioanalysis Meesters
- G P Hooff
- R Guthrie
- A Susi
- G La Marca
- S Malvagia
- A Rosati
Woodford, L. Bioanalysis 2013, 5, 2613−2630.
(3) Meesters, R. J.; Hooff, G. P. Bioanalysis 2013, 5, 2187−2208.
(4) Guthrie, R.; Susi, A. Pediatrics 1963, 32, 338−343.
(5) la Marca, G.; Malvagia, S.; Filippi, L.; Innocenti, M.; Rosati, A.;
- R A Gruters
- T M Luider
Gruters, R. A.; Luider, T. M. Bioanalysis 2012, 4, 2027−2035.
- M Falchi
- S Pellacani
- G Moneti
- R Guerrini
Falchi, M.; Pellacani, S.; Moneti, G.; Guerrini, R. J. Pharm. Biomed.
Anal. 2011, 54, 192−197.
(6) la Marca, G.; Malvagia, S.; Materazzi, S.; Della Bona, M. L.;
- P Denniff
- C Holliman
- L Svensson
- N Weng
- S Patel
Denniff, P.; Holliman, C.; Svensson, L.; Weng, N.; Patel, S.
Bioanalysis 2014, 6, 287−291.
- J Luckwell
- P Denniff
- S Capper
- P Michael
- N Spooner
- P Mallender
- B Johnson
- S Clegg
- M Green
- S Ahmad
- L Woodford
Luckwell, J.; Denniff, P.; Capper, S.; Michael, P.; Spooner, N.;
Mallender, P.; Johnson, B.; Clegg, S.; Green, M.; Ahmad, S.;
Woodford, L. Bioanalysis 2013, 5, 2613−2630.
- R J Meesters
- G P Hooff
Meesters, R. J.; Hooff, G. P. Bioanalysis 2013, 5, 2187−2208.
- G La Marca
- S Malvagia
- S Materazzi
- M L Della Bona
- S Boenzi
- D Martinelli
- C Dionisi-Vici
la Marca, G.; Malvagia, S.; Materazzi, S.; Della Bona, M. L.;
Boenzi, S.; Martinelli, D.; Dionisi-Vici, C. Anal. Chem. 2012, 84,
1184−1188.
- M Barfield
- R Wheller
Barfield, M.; Wheller, R. Anal. Chem. 2011, 83, 118−124.
- C W N Damen
- H Rosing
- J H M Schellens
- J H Beijnen
Damen, C. W. N.; Rosing, H.; Schellens, J. H. M.; Beijnen, J. H.
Anal. Bioanal. Chem. 2009, 394, 1171−1182.
- P E Turpin
- J E C Burnett
- L Goodwin
- A Foster
- M Barfield
Turpin, P. E.; Burnett, J. E. C.; Goodwin, L.; Foster, A.; Barfield,
M. Bioanalysis 2010, 2, 1489−1499.
- R De Vries
- M Barfield
- N Van De Merbel
- B Schmid
- C Siethoff
- J Ortiz
- E Verheij
- B Van Baar
- Z Cobb
- S White
- P Timmerman
de Vries, R.; Barfield, M.; van de Merbel, N.; Schmid, B.;
Siethoff, C.; Ortiz, J.; Verheij, E.; van Baar, B.; Cobb, Z.; White, S.;
Timmerman, P. Bioanalysis 2013, 5, 2147−2160.
- P M M De Kesel
- S Capiau
- W E Lambert
- C P Stove
De Kesel, P. M. M.; Capiau, S.; Lambert, W. E.; Stove, C. P.
Bioanalysis 2014, 6, 1871−1874.
- L Fan
- J A Lee
Fan, L.; Lee, J. A. Bioanalysis 2012, 4, 345−347.
- P Denniff
- N Spooner
Denniff, P.; Spooner, N. Anal. Chem. 2014, 86, 8489−8495.
- M Bosilkovska
- J Deglon
- C Samer
- B Walder
- J Desmeules
- C Staub
- Y Daali
Bosilkovska, M.; Deglon, J.; Samer, C.; Walder, B.; Desmeules,
J.; Staub, C.; Daali, Y. Bioanalysis 2014, 6, 151−164.
- R J W Meesters
- J Zhang
- N A Van Huizen
- G P Hooff
- R A Gruters
- T M Luider
Meesters, R. J. W.; Zhang, J.; van Huizen, N. A.; Hooff, G. P.;
Gruters, R. A.; Luider, T. M. Bioanalysis 2012, 4, 2027−2035.