Volumetric Absorptive Microsampling Technique in the LC-MS Determination of Direct Oral Anticoagulants

Therapeutic drug monitoring (TDM) represents the clinical tool for optimizing individual dosage regimens for a specific patient. In general, TDM is defined as the measurement of clinical parameters which, together with an appropriate interpretation, might directly influence drug prescribing [5]. Through the combination of pharmacodynamics and pharmacokinetics knowledge, TDM enables the assessment of therapy efficacy and individualization of the treatment. TDM is based on the assumption of an exact relationship between dose and drug concentration in the biological matrix (e.g. blood plasma, whole blood, urine, etc.) as well as between concentration and clinical effects [6].

The conventional blood sampling technique is an invasive, tedious, and labour-intensive process. The venipuncture sampling technique is inconvenient and painful, especially for children and critically ill patients [7]. Moreover, trained medical personnel is required to take blood samples. Collected blood samples must be pre-treated as they are not analyzed immediately. This step usually involves centrifugation, aliquoting, freezing, and delivery of samples to a diagnostic laboratory. All mentioned factors increase the risk of errors, are time-consuming, and raise the analysis price [8,9]. Microsampling techniques are a minimally invasive alternative to venipuncture for collecting human body fluids specimens, especially blood samples [7]. Dried blood spot (DBS) sampling is a widely used approach in the research phase of drug development, preclinical toxicokinetic studies, and routine clinical and biochemical examinations. The DBS technique was first introduced to screen phenylketonuria, a metabolic disorder, in the neonatal population in 1963 [10,11]. The sample processing involves using a sample collection card and blood lancet. Briefly, after a finger-prick (or prick of the heel in infants) with the lancet, several drops of blood are absorbed onto the sample collection card, followed by drying, storage, shipping, and extraction steps before the analysis [12]. The shipping and storing conditions are simple and might be performed under ambient temperature and humidity in most applications [13]. Despite many advantages, several limitations arise. First, the vast majority of biochemical and analytical methods require plasma or serum samples; thus, the already existing methods and assays must be redesigned and revalidated specifically for DBS. Next, hematocrit of the blood affects viscosity and may introduce bias in the sampling step, impacting the extraction procedure and, thus, negatively influencing the assay. The spot area on the sampling card typically has a linear, inverse relationship to the blood hematocrit – blood with a high hematocrit level results in a smaller dried blood sample and vice versa [14,15].

During the last two decades, numerous alternatives to DBS have been developed mainly to diminish the hematocrit effect as well as to bring accurate microsampling devices in science and medicine [16]. The new dried blood sampling devices include volumetric absorptive microsampling (the device is called Mitra, produced by Neoteryx, USA), Capitainer qDBS (produced by Capitainer, Sweden), hemaPEN (produced by Trajan Scientific and Medical, Australia), HemaXis DB 10 (produced by DBS System SA, Switzerland), and HemaSpot HF (produced by Spot on Sciences, USA) [17]. In the next section of this review we focused on the volumetric absorptive microsampling technique which is rising in popularity nowadays in the fields of medicine and science.

Volumetric absorptive microsampling (VAMS) is a minimally invasive sampling technique for collecting peripheral blood. The device called Mitra was introduced in the market in 2014 by Neoteryx company (USA). The same year, Denniff and Spooner published a paper describing the VAMS approach using Mitra tips as an alternative microsampling method to DBS [18]. The Mitra tip consists of a hydrophilic polymer fixed to a plastic holder (Figure 1A), allowing the collection of the desired volume by capillary mechanism. Three volumes can be collected depending on the selected tip size – 10, 20, and 30 μL. The sample collection workflow consists of wicking the blood, which takes 3 – 5 seconds, followed by a drying step, usually under ambient room conditions (Figure 1B – D). The storage and transport of the dried tips are recommended in zip-locked bags with desiccant bags [19,20]. Before the analysis, the polymer head of the Mitra tip might be separated, and analytes are extracted with the proper extraction solvent (e.g. methanol, water, acetonitrile, or a mixture of them) [21]. Moreover, the extraction step could also be done in a 96-well autorack and using the liquid handling robots so that the process might be automated [22].

Fig. 1

The collected blood volume is relatively small; thus, the analytical method must be sensitive enough to detect and quantify analytes of interest. Due to the high degree of specificity, sensitivity, and selectivity, the use of liquid chromatography-mass spectrometry (LC-MS) instrumentation is the most popular and suitable technique for analyzing micro volumes of analytes [23]. During the method development, the extracts of Mitra tips might be pre-treated by a procedure like protein precipitation, solid phase extraction, or liquid-liquid extraction methods. The selection of extraction solvent and sample preparation technique is essential as it affects the reduction of matrix effects and extraction recoveries of analytes [24].

The main advantage of VAMS is that it allows hematocrit-independent sample collection with accurate and reproducible blood volumes. However, some authors observed the impact of hematocrit on the extraction efficacy using VAMS [20, 25]. Consequently, optimizing the extraction parameters focusing on the hematocrit range during the method development is recommended. So far VAMS technique has been used in various clinical and nonclinical studies such as pharmacokinetic studies, TDM examinations, or metabolomic studies [16]. The drugs suitable for VAMS microsampling should possess good stability and solubility in water or organic solvents. An example of drugs ideal for TDM using VAMS sampling is summarized in Table 1.

In addition, the introduction of the VAMS technique in routine TDM enables to increase in the adherence of the patients to the therapy, as it allows home sampling without the assistance of trained personnel. Dried Mitra sticks can be delivered to the laboratory, packed in envelopes, and sent via standard postal services [26]. Moreover, several authors using the questionnaire forms confirmed that VAMS home sampling is preferred over traditional blood sampling in the case of routine monitoring [27, 28].

Oral vitamin K antagonist anticoagulants have been used for long-term treatment and prevention of thromboembolic events in many indications for decades [29]. However, due to unpredictable pharmacokinetics and various aspects influencing the efficacy of therapy (mainly genetic factors and food/drug interactions), safer and more effective drug development has arisen [30]. Since 2008, several direct oral anticoagulants (DOACs) have been approved in the EU as a safer alternative to warfarin in avoiding embolic complications. Currently, four DOACs are available in the EU (Figure 2), including direct thrombin inhibitors (dabigatran, the active molecule of prodrug dabigatran etexilate) and direct inhibitors of factor Xa (apixaban, edoxaban and rivaroxaban) [31]. This group of anticoagulants has a rapid onset and offset of action, fewer drug and food interactions, a wider therapeutic window, and a more predictable pharmacological effect than vitamin K antagonists [32]. The basic pharmacokinetics features and drug characteristics of DOACs are summarized in Table 2.

Fig. 2

One of the main advantages over vitamin K antagonists is that DOACs do not require routine biochemical monitoring and dose adjustment. However, their relatively short plasma halftime and once-daily dosing (edoxaban and rivaroxaban) may decrease patients’ adherence to therapy and strongly impact the therapeutic effect [33]. In addition, all of them are metabolized by cytochrome-P450 enzymes, predominantly by CYP3A4 isoform, and interact with P-glycoprotein and breast cancer resistance protein (BCRP). As a consequence, the coadministration of drugs affecting the above-mentioned enzymes and transporters may impact the therapy efficacy. Dose adjustment of DOACs is suggested in some clinical situations like renal failure and during the therapy of extremely obese or thin and elderly patients [34]. As we can clearly observe a growing number of patients taking DOACs, the monitoring of plasma levels might be beneficial and even mandatory in some circumstances. Unfortunately, there is no exact definition of the recommended therapeutic range despite the apparent relation between reached plasma levels and clinical effects [35]. In 2018 International Council for Standardization in Haematology reported recommendations for laboratory measurement of DOACs, where they proposed expected plasma concentrations in patients treated with DOACs in the prevention of stroke, pulmonary embolism, and venous thromboembolism. Nonetheless, it should be mentioned that reference intervals are mainly established on “on-therapy” concentrations [36].

Several specific coagulation assays are established in routine laboratory assessments to monitor plasma levels and perform the TDM of DOACs. They are based on diluted thrombin time, ecarin clotting time, or drug-specific chromogenic anti-Xa tests. However, several disadvantages should be noticed, such as calibration to the particular drug, the imprecise results at low plasma levels, and none of the methods being specific enough for quantifying all DOACs in a single run [37]. Combining liquid chromatography with tandem mass spectrometry (LC-MS/MS) introduces a unique method for measuring DOACs in biological samples. Up to now, several LC-MS/MS methods have been reported to determine DOACs simultaneously in human plasma samples [38–44]. In our laboratory we are finishing the validation of the LC-MS/MS method, allowing high-throughput analysis suitable for routine monitoring. A one-step extraction procedure in 96-well formate was used for sample preparation, enabling to process and analyze more than 80 samples per a working day.

In many indications, direct oral anticoagulants are more often prescribed over vitamin K antagonists. As it was mentioned above, the monitoring of plasma levels might be beneficial in some special situations [1]. Up to now, only one paper has been published for the determination of all DOACs simultaneously using the microsampling technique. Foerster et al. developed and validated LC/MS-MS method using DBS sampling to determine apixaban, edoxaban, rivaroxaban, and dabigatran [45]. Considering the physiochemical properties of DOACs (Table 3), VAMS seems to be a suitable sampling method and might be another possible choice for blood collection. Implementing Mitra stick for LC-MS analysis could help introduce routine TDM of DOACs in broader patient populations. During method development for DOACs determination in our laboratory we also did preliminary experiments using Mitra sticks. Three extraction solvents were evaluated (1% formic acid in 50% methanol, 1% for mic acid in methanol, and methanol) and all of them were capable of extracting all analytes of interest. However, using pure methanol we yielded the highest extraction recovery (data not shown). The aim of our future study will be the implementation of the VAMS technique for the determination of DOACs using the already developed LC-MS/MS method. We will focus on the optimization of extraction procedures (extraction solvent composition, sonication of extracts, time and temperature of extraction), stability of analytes during different storage conditions, and comparison of plasma levels against blood levels.