Introduction

Thromboembolism is a hemostatic disorder associated with a cardiovascular disorder that can cause death (1). It is characterized by the development of thrombus or blood clots in the blood vessels, which can be dislodged, traveled through the blood vessel, and lead to embolism or blockage of a blood vessel (2). Death caused by thromboembolism is commonly associated with coronary artery disease, ischemic stroke related to atrial fibrillation, and pulmonary embolism. In 1856, Virchow postulated a triad of potential causes of thrombosis: stasis, intravascular vessel wall injury, and hypercoagulability (3). Antithrombotic agents can prevent thromboembolism through the inhibition of coagulation (4).

For about 70 years, warfarin has been one of the world's most prescribed anticoagulants. The last decade has seen a significant increase in the prescription of novel direct oral anticoagulants (DOACs) such as apixaban, rivaroxaban, dabigatran, and edoxaban, which is associated with better safety profile and comparable efficacy to warfarin. Nevertheless, there is still an issue that needs to be addressed with the use of DOACs, regarding the limited choice of reversal agents for DOACs (5), the concern of safety in some patients with specific comorbidities (6), and the lack of standardized testing for monitoring of DOACs (7). Although there is a constant decrease in the use of warfarin, along with the discontinuation of the brand name Coumadin in the USA in 2020, it remains the mainstay of anticoagulant therapy in several countries (8, 9).

Evidence of interindividual variations in patients’ responses to the therapy has been observed, causing difficulty in VKAs therapy management. Inadequate coagulation control can lead to episodes of thromboembolism or hemorrhage (10). To prevent this, therapeutic drug monitoring (TDM) is necessary to maintain adequate anticoagulation effects. Prothrombin time (PT) is the conventional test commonly used in therapeutic drug monitoring for patients on VKAs therapy to predict the pharmacodynamic effect and assess the bleeding status of patients. The standard reporting of the PT ratio is expressed as International Normalized Ratio (INR) (11). However, this test is unsuitable for monitoring several instances, such as patient adherence, drug interaction effect, toxicity, and drug resistance. Therefore, it is also essential to ensure that the drug concentration in the bloodstream is appropriate, as another method of TDM (12). This can be achieved by sampling the blood from patients through conventional or microsampling techniques and analyzing the samples using validated bioanalytical methods.

Microsampling is a procedure to collect minute samples (< 100 mL) from a human body for analysis in a minimally invasive manner (13). Although it can be used for any biological fluids, its current application is mostly limited to dried blood samples. There has been an interest in implementing microsampling approaches due to its benefit, which include more convenient sample collection, the ability to obtain samples from pediatric or vulnerable subjects, and a simplified handling process (14). Dried blood spots (DBS) is the most established microsampling approach, which has been used widely for neonatal screening since the 1960s (15). DBS assays have been developed for many medicines over the past decade, such as anticancer drugs (16-18), tacrolimus (19, 20), cardiovascular drugs (21), and antidiabetic drugs (22-24). These last few years also saw the development of volumetric absorptive microsampling (VAMS). This novel dried blood sampling approach enables more accurate and precise blood volume collection than DBS (25). Antibiotics (26), antiseizure medication (27), and paracetamol (28) were among the drugs that have been investigated using VAMS.

We systematically reviews warfarin sampling techniques in human blood, comparing various methods and bioanalytical approaches. No prior systematic review has covered conventional and unconventional sampling techniques for bioanalysis. In this study, records were systematically selected and analyzed.

Methods

Study Protocol

The Preferred Reporting Items for Systematical Reviews and Meta-Analysis (PRISMA) statement is used as a guideline to conduct the literature search (29). For the search strategy, the following descriptors were employed: “warfarin”, “therapeutic monitoring”, “human”, and “chromatography”, using the Boolean operator. The search was conducted in ScienceDirect and PubMed/Medline databases. The potential studies were screened according to the inclusion and exclusion criteria without no restrictions regarding the publication date. A manual search was also conducted using the lists of references from the eligible articles to retrieve additional articles.

Inclusion and Exclusion Criteria

The inclusion criteria for this article review were: (1) Experimental and review studies written in English, (2) Studies that explained sampling techniques and analytical methods of warfarin and its metabolites in human blood samples using the chromatography method.

The exclusion criteria were (1) studies written in other languages besides English, (2) non-related studies, (3) duplicate publications, (4) articles not available as full-text, and (5) studies that also analyze other drugs besides warfarin.

Selection of Studies and Data Collection

All search results were collected and screened. All identified studies are assessed based on the title and abstract without using any specific data extraction form. The data are gathered independently according to the year of publication, study design, bioanalytical method development, and clinical application to patients or human volunteers.

Results and Discussion

A total of 537 potentially relevant studies were identified through database searching. The studies were screened according to the inclusion and exclusion criteria. After the screening process, 9 articles were identified, meeting the inclusion criteria and were considered eligible for full review (30-38). Three articles were retrieved through citation searching from one eligible article (39-41). We have carefully reviewed and used those sources' data to prevent bias. Overall, we concluded that all 12 assessed studies were considered high-quality literature because they met the inclusion criteria and had a low risk of bias. The flowchart of the literature screening process can be seen in Figure 1.

Anticoagulants

Hemostasis is a process the human body undergoes to prevent blood loss during a bleeding episode. The equilibrium condition is maintained between clot formation and obliteration. This process is maintained through interaction involving the cellular components, namely platelets and vascular endothelium, and the coagulation cascade and fibrinolytic system (42).

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A coagulation cascade is a complex model incorporating a sequential activation of proteolytic enzymes, also known as zymogens, and the interlinking of several pathways. The intrinsic pathway depends on contact activation by a negatively-charged surface involving the activation of coagulation factors XII, XI, IX, and VIII. The extrinsic pathway is started with the activation of tissue factor (also known as factor III) during endothelial damage and the activation of factor VII. The resulting end of these two pathways activates factor X, which initiates the common pathway. Factor X, its cofactor (Factor V), and calcium bind to form a prothrombinase complex, which converts prothrombin (factor II) into thrombin (factor IIa), which in turn activate fibrinogen (factor I) into fibrin (factor Ia), and with the help of stabilizing factor (factor XIII) leads to the formation of fibrin crosslink to generate a stable platelet plug/clot (43). Currently, available anticoagulants target one of these pathways.

Warfarin

Warfarin is the most commonly prescribed oral anticoagulant. Originally used as a rodenticide, warfarin was accepted for medical use in 1954 under the brandoumadin (44). Warfarin is a vitamin K antagonist that exerts its effect by competitively inhibiting vitamin K epoxide reductase (VKOR), an enzyme essential for converting vitamin K into an active form. As a cofactor in the activation of coagulation factors II, VII, IX, and X, as well as the coagulation regulatory factors Protein C and S (45), the decrease in activated vitamin K levels can reduce the synthesis of these proteins.

Warfarin is administered as a racemic mixture of R-warfarin and S-warfarin. Warfarin has a half-life of 36 to 42 hours and reaches maximum blood concentration 90 minutes after administration (46). Warfarin is extensively metabolized in the liver, mainly by CYP2C9, and each isomer undergoes a different pathway (47). Intra- and inter-individual variability has been observed during warfarin therapy, causing difficulty in determining the dose regimen for patients. In addition, warfarin also exhibits a narrow therapeutic index. Subtherapeutic and supratherapeutic warfarin dosages may lead to fatal adverse events (48). Patients receiving warfarin are at greater risk for bleeding, and exceeding the therapeutic index may lead to significant hemorrhage. Patients receiving warfarin should be closely monitored to prevent this adverse effect, and the dosage should be tailored accordingly. International Normalized Ratio (INR) is the standard laboratory parameter used to monitor anticoagulant therapy to ensure the safety and efficacy of the therapy (10).

Warfarin is a narrow therapeutic index drug that requires constant monitoring. Typical therapeutic drug monitoring (TDM) requires measuring pharmacokinetic or pharmacodynamic parameters. Selecting a specific pharmacokinetic or pharmacodynamic assay depends on intersubject variability (49). In the case of warfarin, the high variability of pharmacodynamic response necessitates using INR value to monitor the therapy's efficacy and aid the dose adjustment process(50). However, assessment of warfarin concentration in the bloodstream may be beneficial in some clinical situations where sudden and unexpected changes in INR value or patient’s response of unknown origin are observed. In cases such as drug interaction, the measurement of warfarin blood levels may help the physician decide if the increase or decrease of warfarin concentration corresponds to the history of medication and food recently taken by the patient. Warfarin blood levels can also be used to diagnose warfarin intoxication. Warfarin blood level could also be useful in determining patient adherence. Adherence is a major issue with long-term medication regimens (51), particularly requiring constant monitoring, such as warfarin. Self-report results can be unreliable at times, but analysis of warfarin blood level could confirm the result, allowing the physician to take measures to improve patient adherence and increase the therapy's efficacy. This analysis can further differentiate between adherence and resistance, correlating warfarin level to the patient’s response. Resistance can be attributed to acquired factors (drug interaction, non-compliance) or hereditary factors such as genetic polymorphism (52). Measuring the genotype of enzyme polymorphism and phenotype (warfarin and its metabolites concentration) may help the physician assign the patient's metabolizer status (fast, intermediate, poor) and design the algorithm to determine the optimal dose for patients to achieve better efficacy.

["Table", "Table 1. Summary of the results from the literature review.", "9pt", "1"]

Nevertheless, the measurement of warfarin concentration in the blood is seldom used in monitoring. This is possibly caused by the lack of available assays to measure warfarin in blood samples. Developing a bioanalytical method to analyze warfarin in biological samples will enable the use of warfarin blood concentration for monitoring in a routine clinical setting. The method included in this review covered several bioanalytical methods to analyze warfarin in human blood samples.

Blood Sampling Technique

Blood samples from patients or healthy volunteers can be obtained through conventional or micro sampling. The Conventional method is commonly used to collect liquid blood samples such as whole blood, plasma, and serum. Although effective, this method is considered invasive and may be uncomfortable for patients. This leads to the development of a microsampling approach, characterized by a collection of smaller blood volume, which is then stored as dried samples. Dried blood spots (DBS) and volumetric absorptive microsampling (VAMS) are the two methods extensively investigated for clinical application as an alternative to conventional sampling. Table 1 explains samples and sampling techniques used to measure warfarin concentration described in the eligible studies.

Conventional Blood Sampling

The collection of blood samples by phlebotomy is the most common type of biological specimen collection (53). The blood is usually obtained from the median cubital vein in the upper arm, near the radial cutaneous vein. Tourniquet is applied to the arm, and the area will be cleansed with alcohol. Patients are asked to close their hands. After the alcohol has dried, venipuncture is performed with a suitable needle, and once the blood flow begins, patients will be requested to open their hands, the tourniquet released, and the blood will be collected using a tube (54). This method can be applied for plasma and serum sampling, which differs from the sample collection tube. The tube for plasma collection contains an anticoagulant, and the serum tube does not. This method is invasive and may lead to a low recruitment rate in clinical trials, especially pediatric patients. The quality of the phlebotomy will also directly affect the quality of the sample and the analysis result (55). In this review, ten studies have been described to use convenience sampling to analyze human plasma, and 1 study used convenience sampling to analyze serum.

Microsampling

Dried Blood Spots (DBS)

Traditionally used as a neonatal screening sampling method, dried blood spot (DBS) has gained attention as a tool for pharmacokinetic and toxicokinetic studies (56). The first recorded use of DBS was in the early 1960s when Robert Guthrie introduced this method for phenylketonuria screening in newborns using heel prick (15, 57). The blood can be obtained from finger pricks in pediatric and adult patients. The drop of blood is applied to the filter paper, which is then dried for about 2 to 3 hours. The spot is punched after the blood dries for 2 to 3 hours. It can be directly analyzed using suitable analytical techniques or stored and transported to an analytical laboratory before analysis. This method has been developed for other biological matrixes, such as plasma and urine, which are obtained similarly to DBS (58). The limitation of this method is related to the hematocrit (HCT) effect and homogeneity of the area, which may cause bias in the analysis. Although DBS assays have been developed for many medicines over the past decade, such as anticancer drugs (16-18), tacrolimus (19, 20), cardiovascular drugs (21), and antidiabetic drugs (22-24), the author only found 1 study that described the use of DBS for analysis in patients receiving warfarin therapy (59).

Volumetric Absorptive Microsampling

Volumetric Absorptive Microsampling (VAMS) is a method involving the absorption of a liquid sample into a substrate. The device comprises a plastic handler with a hydrophilic porous substrate on the tip (60). The sampling procedure is similar to DBS, in which blood can be directly applied to the tip and dried before being stored or transported for analysis. The volume of the sample absorbed depends on the properties and the amount of substrate, but unlike DBS, VAMS allowed the sample to be measured quantitatively. Another advantage of VAMS is that it can limit the hematocrit effect usually found on DBS samples, leading to different spot sizes on the paper and nonhomogeneity (61). The author finds no article related to using VAMS in the bioanalysis of warfarin.

Bioanalytical Method Development and Validation

Bioanalytical method development aims to define the design, operating conditions, limitations, and suitability of the method for its intended purpose and to ensure that it is optimized for validation. Before developing a bioanalytical method, a complete understanding of the analyte’s properties must be achieved. This includes physicochemical properties, in vitro and in vivo metabolism, protein binding, stability, and other properties that could affect the analytical method. Previous analytical methods already reported could be considered to design a suitable method.

Optimization of the method involves the condition and procedures related to analyte extraction and detection. During this phase, several conditions could be tested, such as the choice and concentration of extraction solvent, flow rate, and detection wavelength, to obtain the optimal bioanalytical method (62).

Bioanalytical Method Validation

Bioanalytical method validation is done to ensure the performance of the optimized method. Validation is usually performed according to guidelines from European Medicines Agency (EMA) (63) or the Food and Drug Administration (FDA) (64). The parameters tested in the validation are listed below:

Selectivity

Selectivity is done to verify that the analytical method can differentiate analyte and internal standard from interference or other components in the sample. Selectivity testing is done by analyzing six individual sources of a blank matrix. The acceptance criteria for selectivity is that the responses from the blank sample are less than 20% from the LLOQ analyte and less than 5% for internal standards (63, 64).

Carry-over

Carry-over has to be detected and minimized in analysis. Carry-over testing is done by injecting a blank sample after the high-concentration sample, calibration standard, or the upper limit of quantification (ULOQ). The acceptance criteria for carry-over is the percentage of carry-over should not exceed 20% of LLOQ. If carry-over cannot be avoided, the sample should not be chosen randomly, and the carry-over effect must be monitored throughout the analysis (63, 64).

Lower Limit of Quantitation (LLOQ)

The lower limit of quantification (LLOQ) is the lowest concentration value of the analyte that can still be calculated accurately and precisely. LLOQ defines the method's sensitivity and should be determined during method development. The LLOQ value of the sample must be five times greater than the blank and not higher than 5% Cmax (63, 64).

Calibration Curve

The calibration curve is the range of concentrations needed to analyze a sample according to the instrument's response. The quantitation range of the analytical method should be established during the method development. The calibration curve must contain at least a blank sample, zero sample (blank + IS), and six concentration levels, including ULOQ as the highest point and LLOQ as the lowest point of the quantitation range. The calibration curve must be reproducible and continuous. A calibration curve must be generated for each analyte if there is more than one analyte. The measured concentration must be within 15% of the actual concentration, except for LLOQ, which should be within 20%. At least 75% of calibration standards must meet these criteria in each run (63, 64).

Accuracy and Precision

Accuracy describes the closeness of the obtained concentration from the analysis with the actual concentration value, expressed in percentage. Precision describes the repeatability of the analyte measurement and is expressed as a coefficient of variation (CV). Accuracy and precision are analyzed using quality control (QC) samples in four concentrations, namely the LLOQ, QCL (three times the LLOQ), QCM (30–50% of calibration curve range), and QCH (75% of the ULOQ). Accuracy and precision must be done in at least five replicates: within-run (single run) and between-run (at least three runs in two days). The acceptance criteria for accuracy is that the concentration must be within 15% of the actual concentration, except for the LLOQ (within 20%). The acceptance criteria for precision validation is the CV must not exceed 15% for the QC sample and 20% for the LLOQ (63, 64).

Dilution Integrity

Dilution of samples should not affect accuracy and precision. Dilution integrity testing is done by spiking the matrix with the analyte concentration above the upper limit of quantification (ULOQ), which then dilutes with the blank matrix. The analysis is done in five replicates for each dilution factor. The acceptance criteria are that the accuracy and precision value must be within 15% (63, 64).

Matrix Effect

The matrix effect is investigated when using mass spectrometry to investigate the effect of ion suppression or ion enhancement on the concentration of the analyte. Matrix effects are tested using a minimum of 6 blank matrixes from 6 individual sources. The matrix factor of the analyte and the internal standard is calculated from the peak area of a spiked matrix with the peak area of the analyte in the standard solution. The testing must be conducted at low and high concentrations (maximum three times LLOQ and close to ULOQ). The acceptance requirement is the CV should not exceed 15% (63, 64).

Stability

Stability testing is carried out to ensure that the process of preparation, analysis, and storage conditions of samples does not affect the concentration of the analyte. Stability must be ensured at every stage of the analytical method. Stability is evaluated using QCL and QCH, which are analyzed immediately after preparation and applied storage conditions. A calibration curve is used for the stability test of QC samples by comparing the obtained concentration with the analyte concentration. The acceptance criteria are that the %diff for each concentration level must not exceed 15%.

The stability condition that must be evaluated is as follows:

1. Stability of the stock solution, working solutions, and internal standard
2. Freeze and thaw stability of the analyte on the matrix
3. Short-term stability of the analyte at room temperature or analysis temperature
4. Long-term stability of the analyte under storage conditions in the freezer

In addition, the following tests could be done if applicable:

1. Stability of the sample at room temperature or storage conditions during analysis
2. Bench-top stability
3. Autosampler stability (63,64).

Most of the studies included in this systematic review have described a full validation of the analytical method. Lv et al. performed validation according to China Pharmacopoeia alongside FDA guidelines. Ghimenti et al. and Lomonaco et al. performed validation according to the IUPAC guideline (65). Because some of the studies were conducted before the publication of FDA guidelines in 2018, validation was conducted according to the guidelines then. Nevertheless, these studies have described a satisfactory validation result. One study only described a standard curve (66). Still, owing to the outdated year of the publication, a method validation guideline had not been established at that time. Two studies did not describe validation, primarily because the studies are developing a novel microextraction method using warfarin as the model drug (32, 67).

["Table", "Table 2. Summary of extraction protocol from the literature review.", "9pt", "1"]