How microsampling is impacting pharmacokinetic and toxicokinetic studies: volumetric absorptive microsampling (VAMS)

ABSTRACT

Microsampling, using minute amounts of biological specimen, is uniquely suited for carrying out human and animal pharmacokinetic and toxicokinetic studies. Indeed, it provides important advantages over common blood-drawing procedures (e.g. increased analyte stability, simpler and cheaper storage and shipping, self-sampling possibility, reduced consumption of materials and animals) while also maintaining similar performance and result reliability. In the microsampling space, volumetric absorptive microsampling (VAMS) is currently considered one of the most important and widespread technologies, thanks to its volume accuracy capabilities, making it natively suitable for quantitative analysis. In this review, an exhaustive treatment of pharmacokinetic and toxicokinetic studies using VAMS is presented, as well as several examples of analytical methods prospectively enabling the employment of VAMS for the same purpose.

1. Introduction

Biological matrix microsampling can be defined as the set of techniques, which allow the reliable sampling of minute amounts of biological matrix, be it liquid, solid or volatile, so that the resulting specimen can be subjected to a suitable analytical workflow with relative ease [1]. What exactly constitutes “minute amounts” can be debated, however generally any sampling technique dealing with matrix volumes (weights) equal to, or lower than, 200 µL (200 mg) is usually considered “microsampling” [2].

In the last few years, microsampling as defined above has emerged as one of the most effective innovations in the field of drug discovery and development [3] and in particular of pharmacokinetic (PK) and toxicokinetic (TK) studies. These studies are characterized by the need to carry out repeated measurements of analyte concentrations in the biological matrix (normally blood, or a blood-derived matrix such as plasma or serum) in a relatively short span of time, and to correlate concentrations with time in the most reliable way possible. Microsampling is highly effective in this respect, since numerous samples can be obtained from the same subject, even when it is a small laboratory animal, without the need to use multiple animals (or greatly reducing the numbers involved) [4]. In particular for TK studies, in many cases microsampling could reduce or eliminate altogether the need for satellite animals (i.e., laboratory animals used specifically for evaluating TK parameters, different from those used for evaluating toxicological properties) [5].

For human subjects, this corresponds to minimal invasiveness, since most microsampling techniques involve the use of a disposable lancet (or of microneedles), producing a simple skin prick. Although microsampling is often better tolerated due to its minimal invasiveness, especially for repeated sampling, some patients may prefer venous sampling, particularly when a central catheter is already in place or when multiple samples can be obtained from a single draw. Moreover, most microsampling techniques involve complete matrix drying, and this in turn usually greatly improves analyte stability, with advantages in terms of sample storage requirements and expenses [6]. Several analytes are stable at room temperature (RT) when the microsample is deprived of water, so that sample shipping and storage can take place without special precautions and with greatly reduced space occupancy (useful for repeated analysis as well). In all respects, these characteristics represent important improvements over traditional sampling methods in terms of sustainability, both from the “3 R” (reduce, reuse and recycle) [7] and the “green analytical chemistry” (GAC) points of view [8–11]. Simple and effortless automatability of procedures is another strong point of microsampling [2].

Indisputably, dried blood spotting (DBS) was the first microsampling technique to become widespread and is still the best known and most applied one [12–14], also in the PK and TK spaces [15]. However, classical DBS, based on the use of cellulose cards on which blood is spotted by simply putting the blood drop into contact with the card (Figure 1), suffers from several shortcomings, the most important one being its lack of volumetric accuracy and hematocrit dependence of sample volume [16]. These make quantitative application problematic. As a consequence, over the years many alternatives have been devised, which are capable of producing volumetrically accurate microsamples, often in the form of dried matrix spots (DMS) [17,18].

Figure 1.

Dried blood spot (DBS) sampling example. Adapted from [10].

One of the first, and most successful, of these alternatives is volumetric absorptive microsampling (VAMS) [19,20]. First commercialized in 2014 under the Mitra® proprietary name, VAMS is based on a device consisting of a plastic handle terminating in a calibrated, porous polymeric tip (Figure 2). The tip, depending on its specific size, is capable of absorbing an accurate volume of matrix (10, 20 or 30 µL). A few seconds are sufficient for completely filling the tip, while drying typically requires about 60 minutes; the latter can be carried out in a dedicated clamshell to protect the sampled device from unwanted contacts and contaminations [21]. Since its introduction, VAMS has known a wide success, with applications in diverse bioanalysis fields, including clinical [22,23], forensic [24], anti-doping [25,26], metabolism [27,28] and indeed PK ones. By combining the practicality of DBS sampling with volumetric accuracy, VAMS greatly enhances the advantages and attractiveness of microsampling for PK and TK studies.

Figure 2.

Volumetric absorptive microsampling (VAMS) example. Adapted from [10].

Despite all these benefits, and despite an indisputable increase in the number of published papers in the field over the years (Figure 3), the total number of scientific articles dealing with microsampling in PKs and TKs remains small, especially if compared to the overall number of papers dealing with these topics but using classical sampling techniques. The reasons for this can be many, however one of the major hurdles is surely the permanence of established workflows in healthcare institutions, and resistance to changing them [16]. Additionally, the replacement of venous plasma with capillary whole blood necessitates investigation into blood-to-plasma concentration ratios, to ensure that pharmacokinetic parameters remain interpretable and comparable to conventional data, and this further complicates adoption.

Figure 3.

Trend of paper publication numbers in the pharmacokinetics (PK) and toxicokinetics (TK) field, in the 2005–2024 period. Source: Scopus, https://www.scopus.com.

Increasing awareness about the benefits, characteristics and performances of microsampling, and in particular of VAMS, in PK and TK studies could help change personnel’s attitude, facilitating the spreading of these techniques and thus sparing unnecessary expenses and animal sacrifices, while still ensuring scientifically sound results. Until now, this task has basically gone unfulfilled, since a literature search of review papers including the “VAMS” term retrieves less than 50 entries, several of them being general reviews on microsampling, and none regarding specifically pharmacokinetics; most of those dealing exclusively with VAMS are concerned with either TDM applications, or the applicability of VAMS to a specific field, which is not pharmacokinetics or toxicokinetics. Just one review paper [19] deals with the use of VAMS in toxicology, but this is mostly concerned with forensic aspects of the technique. One review [29] deals with pharmacokinetic aspects, but only of antimicrobial drugs. Reviews dealing with clinical applications [7] are again mostly focused on TDM and generally with microsampling, not VAMS in particular.

1.1. Aim of the study and methodology

With this review paper, the authors aim to fill a considerable existing gap in general knowledge of the pros and cons of VAMS sampling for the specific purpose of carrying out PK and TK studies, increasing awareness among practitioners and hopefully making those studies more reliable and useful, while at the same time more humane and less expensive as well.

Online literature search was performed at the widest possible temporal scale, i.e., from the oldest available date in each database to the present; although VAMS has been first commercialized in 2014, care was taken not to overlook any early mention of the technique. Three databases were searched, namely Scopus [30], PubMed [31] and Web of Science [32]. Keywords were (VAMS OR volumetric absorptive microsampling) AND (pharmacokinetic OR PK OR clinical); (VAMS OR volumetric absorptive microsampling) AND (toxicokinetic OR TK OR toxicity). Results retrieved through the use of the (microsampling AND pharmacokinetic) and (microsampling AND pharmacokinetic) keywords were also scanned to find any literature related to VAMS, which might have escaped retrieval with the previous search. Keyword location was not restricted, to achieve maximum coverage of the topic.

2. Application of VAMS in PK/TK studies: microsampling as an enabling strategy

VAMS has progressively evolved from a niche technical alternative to a pivotal component in the design of PK and TK studies. Thanks to its capacity to collect low, accurate volumes of whole blood in a user-friendly and minimally invasive way, VAMS offers an operational and analytical paradigm shift, allowing sampling to be moved outside of hospitals and laboratories, reducing the burden on patients and enabling innovative study designs. What emerges from the growing body of literature is that VAMS not only solves the logistical challenges of blood sampling – volume, invasiveness, storage and transport – but also actively expands the scope of PK investigations. This section critically analyses how VAMS has been used across a variety of contexts and drug classes, underlining not only where it has worked, but how and why it has contributed to the success of these studies.

VAMS has shown its strongest justification in pediatric studies, where traditional venipuncture is often impractical or ethically constrained. In a relevant study, furosemide PK was assessed in premature neonates using 10 µL VAMS samples [33]. Despite the vulnerable population, the study achieved a full PK profile, and the plasma – VAMS correlation (r² = 0.962) confirmed its reliability. Similarly, cefepime concentrations in children were determined with a mean bias of − 2.4% vs. plasma and inter-day precision <7% [34]. These results go beyond validation: they demonstrate that PK studies that would be otherwise unfeasible can be realistically carried out using VAMS. This trend is further reinforced by studies in pediatric intensive care units, such as the one on precision dosing of beta-lactam antibiotics including meropenem and cefotaxime [35]. Here, VAMS was integrated into a bedside strategy enabling high-frequency sampling and real-time therapeutic adjustment. While these approaches remain resource-intensive, they signal a clear potential for combining VAMS with clinical decision-making tools.

Importantly, many of these studies explicitly or implicitly rely on bridging strategies, a central concept in modern microsampling research. Plasma-to-VAMS equivalence, often discussed in the context of regulatory guidance [16–21], is now routinely tested using regression models, Bland-Altman analysis and model-based translation (Figure 4). This strategy was effectively demonstrated in a study on dabrafenib and trametinib, where home-collected VAMS samples were translated into serum-equivalent concentrations through a validated population PK model [36] (Figure 5). Another advanced example is the padsevonil development program [37], where more than 2500 paired plasma-VAMS samples were used across four clinical studies to establish a robust population PK model. The FDA endorsed the switch to VAMS-only sampling based on these results, setting a precedent for future model-informed bridging efforts. Ethnic variability was explored in a study comparing VAMS and conventional sampling for padsevonil in Chinese volunteers [38], showing that drug exposure was comparable among ethnicities and supporting the transferability of Western PK data. This concept was further extended to large molecules in a Phase I PK study on monoclonal antibodies, where VAMS was applied in both rhesus monkeys and healthy volunteers, using venous and fingerstick samples in parallel with serum references [39], highlighting its potential in large-molecule applications and early-phase research.

Figure 4.

Agreement between paracetamol concentrations in VAMS and conventional matrices. (a) Bland-Altman plot comparing cVAMS and blood concentrations of paracetamol; (b) passing-bablok regression analysis of paracetamol concentrations in vVAMS plotted against blood concentrations; (c) boxplots of paracetamol cVAMS/vVAMS concentration ratios per sampling time point. Data illustrate acceptable bias and consistent correlation between microsampling and traditional methods. Adapted from [68].

Figure 5.

Development of a population pharmacokinetic (popPK) model-informed VAMS-to-serum conversion model. PopPK models were used to generate serum maximum a posteriori (MAP) estimates from VAMS for dabrafenib (a) and trametinib (b). Adapted from [36].

A similar strategy was applied in the global pediatric study of baricitinib [40], where VAMS replaced plasma in a sparse sampling design, maintaining model robustness. These studies illustrate a clear point: when VAMS is not simply validated analytically, but embedded into the design logic of the study, it becomes more than a replacement – it becomes enabling.

Immunosuppressants are particularly well represented in this context. In a drug-drug interaction study with tacrolimus and fluconazole, VAMS sampling showed good correlation with plasma (R² = 0.994, bias 4.6%) in adult kidney transplant recipients [41]. Such studies stress an important theme echoed in the literature: while PK and TDM have different endpoints, the analytical robustness required is often the same and VAMS can meet it.

The potential of VAMS extends beyond clinical sampling. In preclinical models, serial microsampling with VAMS has reduced the number of animals required. Tacrolimus in rats (Figure 6) [42] and estetrol in rodents [43] were successfully monitored with full PK coverage, aligning with animal welfare principles and reducing biological variability across groups.

Figure 6.

Representative chromatograms of tacrolimus in VAMS extracts and wet blood samples. (a) Blank rat blood in VAMS extracts, (b) tacrolimus at the low-quality control level (0.6 ng/mL) in VAMS extracts, (c) blank in rat wet blood, (d) tacrolimus at the low-quality control level (0.6 ng/mL) in rat wet blood. Adapted from [42].

VAMS has also been instrumental in expanding PK research in oncology. The ability to monitor capecitabine [44] and nab-paclitaxel [45] using microsamples enabled the evaluation of exposure-toxicity relationships in outpatient settings, with limited disruption. These studies signal that VAMS is not just acceptable but preferable when analytical needs and operational complexity are balanced.

Innovative analytical techniques have further broadened the application of VAMS. Oestrogens were extracted from 10 µL VAMS using a nano-LC protocol with sub-ng/mL sensitivity, without derivatization [46]. Polyphenol metabolism was tracked in nutritional PK with time-course exposure using VAMS [47], highlighting its suitability for labile or unstable analytes.

In the context of cannabinoids, the application of VAMS in pediatric patients for THC and its metabolites [48] demonstrated consistent accuracy across a wide hematocrit range. This is significant, given that hematocrit bias is often seen as a limitation of microsampling [49] and controlling it remains a defining aspect of method validation. Although VAMS exhibits reduced hematocrit dependency compared to DBS, hematocrit assessment may still be relevant in specific applications, particularly when working with vulnerable populations or extreme hematocrit values. Field studies demonstrated that VAMS allows PK and TDM research to be conducted even in resource-limited environments, as long as temperature, drying time and sample handling are properly managed [50].

Additional examples consolidate this picture. Paracetamol quantification using VAMS in a clinical PK study confirmed its suitability for deriving full PK profiles in hospitalized patients [51] and using VAMS for vincristine PK in pediatrics [52] all lead to the same conclusion: VAMS allows for complex, high-resolution PK data collection in settings previously inaccessible to traditional workflows. Midazolam PK in critically ill children were successfully profiled from VAMS-collected whole blood in a study comparing it to standard wet sampling [53]. In a crossover study on tranexamic acid, VAMS was used to profile the drug after intravenous, intramuscular, and oral administration, with complete PK curves generated in healthy volunteers [54]. A preclinical study investigated the TK of paracetamol in rats using dry and wet VAMS as well as diluted whole blood, with time-resolved sampling and full TK parameter evaluation [55].

Matrix comparison and equivalence assessment remain key in all these studies. Whether through direct statistical comparison or model-based extrapolation, researchers continue to validate VAMS not just as a tool of convenience, but as a valid analytical matrix [56]. When properly controlled, bias is generally within 10–15% and precision matches bioanalytical expectations [29]. Relevant applications of VAMS in PK and TK studies are summarized in Table 1.

Table 1.

Representative VAMS applications in PK and TK studies.

Analyte(s)	Biological matrix	Sample volume (µL)	Pretreatment	Analysis	PK/TK parameters	Study population/model	Matrix comparison/bridging	Notes	Ref.
Furosemide	Whole blood	10 µL	Protein precipitation (PPT)	LC-MS/MS	Cmax, AUC, Tmax	Pediatric patients (premature neonates)	Plasma-VAMS bridging in neonates (paired sampling)	PK profiling in neonates using VAMS	[28]
Cefepime	Whole blood	10 µL	UAE, dilution	LC-MS/MS	AUC	Pediatric patients	Method validated against plasma samples	Validation included hematocrit effect assessment	[19]
Cefotaxime, ceftriaxone, cefepime, meropenem, vancomycin, piperacillin	Whole blood (critically ill children)	10 µL	Evaporation, reconstitution	LC-MS/MS	Cmax, AUC, trough concentrations	Critically ill pediatric patients	Paired VAMS-plasma for precision dosing in children	Precision dosing in pediatric intensive care	[29]
Dabrafenib, Trametinib	Whole blood (capillary, self-sampled)	20 µL	Protein precipitation	LC-MS/MS	AUCτ, Cmin, CL/F (popPK-derived)	Adult patients with BRAFV600-mutant melanoma	VAMS – serum equivalence via population PK model	Home sampling; individual parameter estimation from at-home VAMS	[32]
Tacrolimus	Whole blood (VAMS)	10 µL	UAE, centrifugation	LC-MS/MS	AUC, Cmax	Adult kidney transplant recipients	Sparse sampling with PopPK model (VAMS vs plasma)	Model-based dose adjustment strategy	[33]
Padsevonil	Whole blood	10 µL	UAE, reconstitution	LC-MS/MS	Cmax, AUC, Tmax	Healthy Chinese volunteers	Ethnic PK comparison: VAMS vs historical plasma data	Ethnic sensitivity evaluation	[34]
mAb1	Whole blood (VAMS), serum	20 µL	Pellet digestion	LC-MS/MS	PK profile in NHP and healthy volunteers	Rhesus monkeys and healthy subjects	VAMS (fingerstick and venous) vs serum	Bridging study; impact of anticoagulant (EDTA) on IS response	[35]
Baricitinib	Whole blood	10 µL	Evaporation, reconstitution	LC-MS/MS	Cmax, AUC, half-life	Pediatric patients (2 large global studies)	Bridging study VAMS-plasma in global pediatric trials	Large-scale pediatric clinical validation	[16]
Tacrolimus	Whole blood	10 µL	UAE, evaporation, reconstitution	LC-MS/MS	AUC, Cmax	Adult renal transplant patients	VAMS vs plasma for DDI in renal transplant recipients	Study focused on drug-drug interactions using VAMS	[77]
Tacrolimus	Whole blood (rat)	20 µL	Extraction, evaporation, reconstitution	LC-MS/MS	Cmax, AUC	Rats (in vivo study)	Rat study: VAMS vs wet blood and plasma	Microsample applicability in rodent model	[21]
Estetrol (E4)	Whole blood	10 µL	Protein precipitation, centrifugation	LC-MS/MS	Cmax, AUC	Mouse model	Preclinical model (mouse): VAMS vs plasma data	Feasibility of preclinical microsampling	[36]
Capecitabine, 5-FU	Whole blood (VAMS)	20 µL	Evaporation, reconstitution	LC-MS/MS	Cmax, Tmax, AUC	Cancer patients (adjuvant chemotherapy)	Proof-of-concept comparison VAMS vs venipuncture	Outpatient-friendly sampling in oncology	[37]
Nab-paclitaxel	Whole blood	10 µL	UAE, evaporation, dilution	LC-MS/MS	Cmax, AUC, Tmax	Cancer patients	Whole blood/plasma correlation tested	Correlation analysis of matrices	[38]
Estradiol, estrone, ethinylestradiol	Whole blood	10 µL	Nanofluidic extraction and LC-MS/MS	LC-MS/MS	AUC, Cmax	Healthy women (clinical study)	Nano-LC bridging vs conventional LC for estrogen levels	Nano-extraction implementation	[39]
Polyphenol metabolites (e.g., ferulic acid, p-coumaric acid)	Whole blood	20 µL	UAE, centrifugation	LC-MS/MS	AUC, Tmax	Healthy volunteers after barley biscuit intake	Nutritional PK study, VAMS vs plasma comparison	Nutritional bioavailability and metabolite tracking	[40]
Δ9-THC, CBD, 11-OH-THC	Whole blood (VAMS)	10 µL	Evaporation, reconstitution	LC-MS/MS	Cmax, Tmax, AUC	Pediatric epilepsy patients	Matrix comparison with plasma for cannabinoids in children	Cannabinoid PK in vulnerable population	[41]
Paracetamol, paracetamol glucuronide, paracetamol sulfate	Whole blood	10 µL	Evaporation, protein precipitation	LC-MS/MS	Cmax, AUC	Hospitalized patients	Matrix effect evaluated in VAMS vs wet blood	Focused on matrix effect quantification	[44]
Vincristine	Whole blood (pediatric patients)	10 µL	UAE, reconstitution	LC-MS/MS	AUC	Pediatric cancer patients	Clinical use of VAMS validated against venipuncture	Therapeutic monitoring in pediatric cancer	[45]
Midazolam, 1-OH-midazolam	Whole blood (wet and dried VAMS)	10 µL	UAE, protein precipitation	LC-MS/MS	Cmax, AUC, half-life	Pediatric patients in intensive care	Wet vs dry VAMS samples (within-method comparison)	Drying effect assessment on sample stability	[46]
Tranexamic acid	Whole blood	10 µL	UAE, centrifugation	LC-MS/MS	Cmax, AUC	Healthy volunteers	Comparison between VAMS, capillary and venous samples	Evaluation of alternative sampling routes	[47]
Paracetamol	Whole blood (VAMS)	10 µL	Protein precipitation	LC-MS/MS	AUC₀–₂₄, AUC₀–₇, Cmax, Tmax	Wistar Han rats	Dry VAMS vs wet VAMS vs diluted whole blood	Toxicokinetic profiling in preclinical model	[48]
Padsevonil	Whole blood	20 µL	UAE, evaporation, dilution	LC-MS/MS	Cmax, Tmax, AUC	Healthy volunteers and epilepsy patients	Direct comparison VAMS vs plasma (healthy + patients)	Integrated clinical PK bridging strategy	[49]

Taken together, the studies reviewed in this section reveal a coherent trend: VAMS is no longer experimental. Its application in PK and TK is grounded in analytical rigor, supported by appropriate validation strategies and increasingly embedded into protocol design. Rather than simply replacing plasma or DBS, VAMS is enabling new approaches – in patient populations, in decentralized models and in sample-limited settings. The key challenge ahead will not be whether VAMS works, but how to optimize its implementation for each analytical and clinical context.

3. Methodological frameworks and analytical foundations for PK/TK studies with VAMS

A considerable portion of the literature on VAMS is dedicated not to direct PK or TK studies, but to the development, validation and technical evaluation of methods that can enable or support such applications. These studies, while not exploring concentration-time relationships or model-based PK analysis, often deal with therapeutic drug monitoring, analyte stability, bridging with conventional matrices, and sample handling – providing a solid framework for future PK/TK deployment.

In a multi-analyte method development for opioids and their metabolites, VAMS was validated against both venous blood and plasma to support future PK applications, though no time-course or PK parameters were derived [57]. Mitotane therapy monitoring is a representative example, where VAMS offered improved sample stability in dried format and simplified logistics [58]. Further examples include dried microsampling protocols validated for antipsychotics and antidepressants, showing adequate stability and matrix correlation for possible longitudinal use [22,23,59]

Among these, several works focused on the development and validation of bioanalytical methods suitable for VAMS sampling in clinical contexts. For example, robust protocols were developed for tyrosine kinase inhibitors [60], and trikafta [61], each including assessments of selectivity, accuracy, and precision. Studies involving antibiotics [62] similarly provided full validation data, often complemented by matrix comparison or hematocrit evaluations. Another study focused on oncology patients, demonstrating that multiplex assays including TDM-relevant drugs can be effectively validated for VAMS [63]. Additional analytical frameworks have also been developed for drugs of abuse and doping agents, with full validation on VAMS and DBS platforms and applications across forensic and sports medicine contexts [24,64,65]

While these methods were not applied to study drug disposition over time, they addressed key analytical variables relevant to kinetic studies [66]. These aspects are central when extending VAMS workflows into longitudinal designs, particularly in decentralized or at-home sampling scenarios. Innovative preconcentration strategies have also been explored. Cenobamate monitoring in epilepsy patients using VAMS demonstrated how single-point therapeutic drug monitoring can be implemented in a real-world clinical setting [67]. A complementary study validated VAMS-based metabolite profiling for paracetamol and its major pathways, with a view to future PK applications [68] while one study optimized electromembrane extraction for paracetamol in VAMS samples [69], demonstrating the potential of selective enrichment techniques for future PK workflows.

Several papers addressed practical or logistical dimensions, simulating real-world challenges. For instance, studies evaluated analyte stability under varied temperature and storage conditions, or VAMS applicability during extended transport and under environmental stress [70,71]. These contributions are highly relevant for the design of PK/TK studies in outpatient, home-based, or multicentric settings [3]. A study on voriconazole quantification [72] demonstrated strong plasma agreement and stability, confirming its potential for routine clinical use. Equally relevant are those studies that aimed at bridging between matrices [73,74]. compared VAMS-derived concentrations to those from plasma or whole blood. These data sets help clarify how microsample-based concentrations relate to standard bioanalytical references.

Moreover, a recent study applied VAMS to quantify tryptophan-related biomarkers in mouse blood, supporting future integration in preclinical TK studies [26]. A stability-oriented study on trastuzumab [75] confirmed the applicability of VAMS to therapeutic proteins, demonstrating its stability and potential compatibility with biologics, as supported by stability-focused investigations. Although framed in a forensic or regulatory setting, these data highlight VAMS compatibility with low-concentration analytes and complex matrices – features directly applicable to TK study designs. The ability to move sampling out of hospitals is another recurring theme. A centralized monitoring workflow for oncology patients using VAMS was proposed and validated for logistical feasibility [76].

Taken together, these studies form the analytical and operational backbone for expanding VAMS into full-scale PK and TK investigations [2]. Their collective evidence, supported by recent reviews and methodological analyses, shows that the transition from technical feasibility to routine kinetic application is not only possible, but increasingly well justified [11]. A selection of validated methods with potential for PK/TK implementation is presented in Table 2.

Table 2.

VAMS-based methods for PK/TK-oriented workflows.

Analyte(s)	Biological matrix	Sample volume (µL)	Analytical scope/application	Potential for PK/TK use	Matrix comparison	Notes	Ref
Clozapine and metabolites	Whole blood (b-VAMS), plasma (p-VAMS)	20 µL	TDM in psychiatric patients treated with clozapine	Limited – no time-resolved data	b-VAMS and p-VAMS vs classical plasma	VAMS and mfDBS validated; applied to real patient samples	[21]
Cocaine and metabolites	Capillary blood (b-VAMS)	20 µL	Analytical validation and application to forensic samples	Limited – no time-course, post-use	b-VAMS vs venous blood and DBS	Focus on forensic scenarios, method validated and applied	[23]
Opioids (multiple)	Whole blood (VAMS, DBS)	10 µL	Comparison of DBS and VAMS for opioid TDM	Potential – method adaptable	VAMS vs DBS	Validated protocol, useful for decentralized TDM	[51]
Mitotane	Whole blood (VAMS)	20 µL	TDM in adrenocortical carcinoma patients	Limited – no time-course data provided	VAMS vs plasma	Cross-sectional study; potential for dose individualization	[52]
Sertraline, Fluoxetine, Citalopram, Vortioxetine and metabolites	Whole blood (VAMS), oral fluid (VAMS)	20 µL	TDM in patients with depression and related disorders	Limited – no time-course data, cross-sectional only	VAMS vs conventional matrices (plasma, OF)	Microsampling validated and applied to patient samples	[53]
Imatinib	Whole blood (VAMS)	20 µL	Quantitative method for clinical TDM in oncology	Limited – TDM without time-course sampling	VAMS vs venous blood	Validated for stable levels in patient follow-up, no PK profile	[54]
Ivacaftor, Tezacaftor, Elexacaftor	Whole blood (VAMS)	10 µL	Quantitative method for cystic fibrosis therapy monitoring	Potential – if integrated into clinical trial designs	VAMS vs venous blood	Validated against plasma; applied in real patients	[55]
Amoxicillin, Ampicillin, Meropenem, Cefadroxil	Whole blood (VAMS and DBS)	10 µL	Comparative method validation for antibiotics quantification	Yes – supports TDM and future PK with clinical sampling	VAMS vs DBS	Full validation with HCT and drying conditions assessment	[56]
10 Tyrosine kinase inhibitors	Whole blood (VAMS)	20 µL	Multiplex LC-MS/MS method for TDM	Potential – if integrated into time-course designs	Plasma vs VAMS in clinical samples	Validated across large dynamic range, but no PK application yet	[29]
Oxycodone and metabolites	Whole blood (b-VAMS, DBS), urine	20 µL	Bioanalytical method for TDM, forensic and anti-doping purposes	No – stability and validation only	VAMS vs DBS vs urine	Focus on matrix effect, extraction and stability comparison	[57]
THC and metabolites, SCRAs	Whole blood (VAMS), DBS	20 µL	Screening after cannabis use	Limited, no defined timepoint	VAMS vs DBS and plasma	Real exposure but no time-profile or PK	[58]
Paracetamol	Whole blood	20 µL	Proof-of-concept for finger-prick sampling method	Limited – no PK study performed	VAMS vs DBS vs plasma (partially)	Focus on self-sampling and feasibility	[59]
Cenobamate	Whole blood (VAMS)	10 µL	Therapeutic drug monitoring in epilepsy	Limited – no time-profile design	None	Focused on treatment compliance	[60]
Paracetamol and metabolites	Whole blood (cVAMS and vVAMS)	10 µL	Method validation and metabolite profiling	Potential – no time-course	VAMS vs. venous/DBS	B:P ratio, HCT effect, no PK design	[61]
Paracetamol	Whole blood (VAMS)	20 µL	Electromembrane extraction optimization	Yes – method adaptable to PK sampling	Plasma vs VAMS (preliminary)	Selective preconcentration strategy evaluated	[62]
Hydroxyurea	Whole blood (VAMS)	10 µL	TDM in sickle cell disease	Limited – no time-course design	Not reported	Monitoring trough levels for adherence	[63]
Miltefosine	Whole blood (VAMS)	10 µL	Bioanalytical method for neglected diseases	Yes – validated method for field studies	VAMS vs plasma (preliminary)	Field-adaptable; suitable for clinical PK designs	[64]
Meropenem	Whole blood (VAMS)	10 µL	Quantification of meropenem in human whole blood using VAMS	TDM study in clinical setting; method could support PK profiling	Comparison with plasma (good agreement shown)	High stability; method validated for hospital implementation	[66]
Everolimus	Whole blood (VAMS)	10 µL	TDM in oncology setting	Limited – used for exposure monitoring	VAMS vs plasma	Implemented in hospital workflow, not for PK studies	[67]
Alternaria mycotoxins	Whole blood (VAMS)	10 µL	Biomonitoring study in exposed population	Low – no time-resolved data	None	Exposure study, not focused on kinetics	[68]
Tryptophan derivatives	Whole blood (VAMS)	20 µL	Method validation for biomarker quantification	No – cross-sectional design only	No – cross-sectional design only	Wide biomarker panel, absolute quantitation, no time-course.	[69]
Trastuzumab	Whole blood (VAMS)	10 µL	Stability of monoclonal antibodies in VAMS	Potential – not explored in the study	None	Focus on analyte stability; no patient samples	[70]

4. VAMS-plasma conversion strategies

Several studies have addressed the conversion of VAMS-derived concentrations into plasma equivalents, primarily because clinical interpretation frameworks (such as therapeutic ranges, PK reference intervals, and exposure thresholds) have traditionally been established using plasma data. For example, in therapeutic drug monitoring (TDM), reference values are typically defined in plasma, necessitating a matrix translation when using whole blood microsamples. These conversions are molecule-specific and depend on the analyte’s distribution characteristics. To bridge this gap, various strategies have been adopted in the literature, including the use of blood-to-plasma ratios, empirical correction factors, or direct comparison of VAMS and plasma concentrations. Explicit correction strategies were applied several studies [23,24,38,59,64,65,68,70], while high correlation between VAMS and plasma levels (even in the absence of formal correction) was reported [37,53,60]. Additional comparative evaluations across matrices, often for PK profiling or stability assessment, were also carried out [36,55,72].

These examples reflect a transitional phase in the application of whole blood microsampling. As long as plasma remains the clinical reference matrix, such conversions are often necessary. However, with growing acceptance of volumetric microsampling in regulated bioanalysis, one may envisage a future where therapeutic ranges and clinical targets are established directly in whole blood matrices – potentially making the need for plasma-based correction obsolete.

5. Future perspective

VAMS is uniquely placed to take advantage of current and future developments in the field of PK and TK. Since it has the same advantages as DBS, but with the added benefits of volumetric accuracy and hematocrit independence, in the next 5–10 years it will probably reach and overtake older (albeit more entrenched) microsampling techniques in terms of use, availability and diffusion. Not just this, the continued evolution and enhancement of analytical technique performances will also mean that VAMS will mostly be equally effective and reliable, and thus equally applied, as classical biosampling techniques, with corresponding important savings in terms of material expenses and animal lives. Larger, statistically more powerful PK and TK studies will be made feasible, with consequent important benefits regarding their accuracy and reliability. However, the higher unit cost of VAMS devices compared to DBS cards may represent a limiting factor, particularly in large-scale or resource-limited studies, and should be weighed against their analytical and logistical benefits. Finally, enhanced greenness, environmental friendliness and overall sustainability of PK and TK studies will be close at hand and indeed made possible and economically viable by the application of VAMS.

Funding Statement

The Authors M.P., R.M. and L.M. acknowledge the financial contribution from Alma Mater Studiorum - University of Bologna through Fundamental Oriented Research (RFO) funds. H.M.S., C.L. and J.L.C would like to thank the Associate Laboratory for Green Chemistry - LAQV which is financed by national funds from FCT/MCTES ([LA/P/0008/2020] DOI 10.54499/LA/P/0008/2020, [UIDP/50006/2020] DOI 10.54499/UIDP/50006/2020 and [UIDB/50006/2020] DOI 10.54499/UIDB/50006/2020), as well as the PROTEOMASS Scientific Society (Portugal) for funding support [General Funding Grant 2024–2025].The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Article highlights

• Microsampling offers important advantages for pharmacokinetic (PK) and toxicokinetic (TK) studies

• Volumetric absorptive microsampling (VAMS) is one of the most established and reliable techniques in this space

• PK and TK applications of VAMS have been diverse, highly effective and top performing

• Currently published innovative methods are taking advantage of VAMS for prospectively enabling future PK and TK studies

• Increased use of VAMS in PK and TK could have a strongly positive sustainability impact

Author contributions

Michele Protti: Methodology, Data curation, Writing – original draft, Writing – review & editing; Roberto Mandrioli: Conceptualization, Writing – original draft, Writing – review & editing; Hugo M. Santos: Visualization, Writing – review & editing; Carlos Lodeiro: Methodology, Writing – review & editing; José L. Capelo-Martínez: Methodology, Writing – review & editing; Laura Mercolini: Conceptualization, Supervision, Methodology, Visualization, Writing – review & editing, Supervision.

Disclosure statement

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Reviewer Disclosure

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.