Microsampling Assays for Pharmacokinetic Analysis and Therapeutic Drug Monitoring of Antimicrobial Drugs in Children: A Critical Review.

Abstract

Purpose:

With the increasing prevalence of multi-drug resistant organisms, therapeutic drug monitoring (TDM) has become a common tool for assuring the safety and efficacy of antimicrobial drugs at higher doses. Microsampling techniques, including dried blood spotting (DBS) and volumetric absorptive microsampling (VAMS), are attractive tools for TDM and pediatric clinical research. For microsampling techniques to be a useful tool for TDM, it is necessary to establish the blood–plasma correlation and the therapeutic window of antimicrobial drugs in the blood.

Methods:

DBS involves the collection of small volumes of blood (30 – 50 μL per spot) on a filter paper, while VAMS allows the accurate and precise collection of a fixed volume of blood (10–30 μL) with microsampling devices. One of the major advantages of VAMS is that it reduces or eliminates the volumetric blood hematocrit (HCT) bias associated with DBS. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) is a powerful tool for the accurate quantification of antimicrobial drugs from small volumes of blood specimens.

Results:

This review summarizes the recent LC-MS/MS assays that have employed DBS and VAMS approaches for quantifying antimicrobial drugs. Sample collection, extraction, validation outcomes, including the inter- and intra-assay accuracy and precision, recovery, stability, and matrix effect, as well as the clinical application of these assays and their potential as tools of TDM are discussed herein.

Conclusion:

Microsampling techniques, such as VAMS, provide an alternative approach to traditional plasma sample collection for TDM.

Introduction

Antimicrobials are the most common types of medications prescribed to hospitalized children, especially to those who are critically ill. Point prevalence studies have estimated that nearly half of the infants in neonatal intensive care units and three-quarters of children in pediatric intensive care units are prescribed at least one antimicrobial drug on a daily basis during hospitalization¹. Antimicrobials comprise the most common class of medications that cause adverse drug events in hospitalized children². Although several of the toxicities associated with the use of antimicrobials are dose-dependent^{3, 4}, therapeutic drug monitoring (TDM) is routinely performed for only a few select agents. With the increasing frequency of infections due to drug-resistant bacteria and the escalation in the dose of drugs, the provision of personalized antibiotic dosing for achieving targeted drug concentrations has become more important than ever for maximizing the safety and efficacy of these agents, while minimizing the emergence of resistance. Traditional TDM of antibiotics involves the quantitative measurement of drug concentrations in the serum or plasma, collected at specific intervals during dosing regimens. Most often, both the peak concentrations and trough concentrations or only the trough concentrations are determined for drugs such as vancomycin and aminoglycosides^5–7 for TDM in children.

As infections due to drug-resistant bacteria are becoming more prevalent, achieving the effective drug concentration is of paramount importance. Apart from simply monitoring drug concentrations for assessing the risks of toxicity, TDM can also be used to ensure that the optimal drug concentration associated with clinical efficacy is achieved. In combination with advanced pharmacometric tools, the measurement of drug concentrations can aid personalized antibiotic dosing and to attain therapeutically effective drug concentrations. TDM can provide information regarding the dosing of agents that cannot be subjected to traditional TDM owing to toxicity, including β-lactams, triazole antifungals, and fluoroquinolones. There is a definite need for the wider implementation of antimicrobial TDM in patients with large inter-individual variability in drug pharmacokinetics (PK), including critically ill infants and children⁸, or obese individuals. A representative schematic describing the pharmacodynamic target for the β-lactam antibiotic, cefepime, is depicted in Figure 1.

Figure 1. Pharmacodynamic targets for antimicrobial therapy.

Concentration-time profiles for two hypothetical patients with the same volume of distribution but different total body clearance and receiving the same dose of cefepime. For both the patients, a minimum inhibitory concentration (MIC) of 4 μg/mL was used, which is considered susceptible and dose-dependent by the Clinical and Laboratory Standards Institute. In both the patients, the concentration of the drug was maintained above the MIC (hashed line) for >60% of the dosing interval. The circle designates the time at which the concentration of the drug for patient B falls below the MIC (7.6 h; free drug concentration greater than the MIC [fT > MIC] for 95% of the dosing interval). The drug concentrations for patient A were maintained above the MIC for the entire dosing interval (100% fT > MIC). Only in patient A (blue line), the drug concentrations were maintained 4-fold above the MIC (horizontal dotted line) for at least 60% of the dosing interval. The bactericidal activity of β-lactam antibiotics, including cefepime, increases by nearly 4 times the MIC, and therefore this pharmacodynamic target is often used to define the optimal inhibitory threshold for these agents. X marks the time at which the drug concentration for each patient falls below 4 times the MIC: patient A achieved fT > 4x MIC for approximately 80% (6.3 h) and patient B achieved and maintained fT > 4x MIC for approximately 56% (4.5 h) of the interval.

C_max, maximal concentration (peak); C_min, minimal concentration (trough); MIC, minimum inhibitory concentration

In this regard, the use of an universal, weight-based dosing strategy is suboptimal in children, particularly in those who are critically ill. Owing to the scarcity of pharmacokinetic/pharmacodynamic (PK/PD) data in children, clinicians frequently need to perform TDM for optimal treatment. Augmented renal clearance in critically ill children with sepsis impedes the achievement of target concentrations⁹, which necessitates the use of higher doses of antibiotics for serious infections¹⁰. However, the empiric use of exceedingly large doses induces toxicity, requiring clinicians to weigh the risks of inefficacy and toxicity when selecting an empiric dosing strategy.

In order to facilitate the TDM of antimicrobial drugs in children, quantitative drug assays must provide accurate and precise results with a small sample volume in a short time. For antimicrobial drugs, the concentrations are typically measured from plasma or serum samples using validated high-performance liquid chromatography (HPLC) or liquid chromatography-tandem mass spectrometry (LC-MS/MS)^11–14. The dried blood spot (DBS) and volumetric absorptive microsampling (VAMS) methods are attractive alternatives to traditional plasma collection owing to the following reasons: 1) they allow for the collection of small blood volumes that are within the safe limits of blood collection; 2) they are less invasive, and can therefore alleviate stress due to venipuncture blood withdrawal; 3) they reduce the risk of infection, including central-line associated bloodstream infections, that are common in critically ill pediatric patients in intensive care units¹⁵; 4) they eliminate visits to the hospitals or clinics, allowing for sample collection at home; 5) sampling can be done at a convenient time, without the patient having to depend on a phlebotomy team, and 6) they allow for the collection of samples at multiple timepoints since blood withdrawals are minimal. However, the correlation between microsampling (DBS or VAMS)-based approaches and traditional sampling approaches (plasma or serum) needs to be established. The purpose of this critical review is to systematically discuss the recently reported LC‐MS/MS methods that utilize DBS and VAMS for quantifying antimicrobial drugs, along with information on sample collection, sample preparation, assay validation, and clinical application. Further considerations for the future implementation of microsampling-based LC‐MS/MS assays for TDM will be discussed.

Sample Collection

DBS

DBS is a microsampling technique that utilizes filter paper, including Whatman DMPK-A, DMPK-B, and DMPK-C cards, for collecting small volumes of whole blood (30 – 50 μL per spot) for quantitative analysis. Sample collection is generally performed in two ways: 1) by a finger or heel stick, following which the sampling site is pressed onto the designated circle on the DBS paper for collecting whole blood, or 2) an exact volume of whole blood is pipetted onto the card¹⁶. DBS allows the collection of samples from remote locations owing to the ease of sample shipping and storage. However, DBS samples are known to have limitations in drug quantification owing to sample heterogeneity, hematocrit (HCT) variance, drying time, and analyte recovery^{17, 18}. Sample heterogeneity is a known issue with DBS sampling. Patients have varying HCT values depending on their gender, age, and health conditions. Whole blood samples are thinner and spread more easily on DBS paper when the values of HCT are lower. The opposite is observed at higher values of HCT, since the samples of whole blood are thicker. There are several validated methods for quantifying antimicrobial drugs by DBS-based LC-MS/MS methods, which have been applied for the analysis of clinical samples^{17, 19–21}.

VAMS

Similar to DBS, the VAMS sampling technique enables the collection of 10 – 30 μL of whole blood (Mitra® device®, Neoteryx, LLC, Torrance, CA). The device collects an accurate volume of sample, and reduces or eliminates the issues with sample heterogeneity and HCT variability described for DBS, where the blood spreads differently on the filter paper owing to differences blood viscosity^{17, 22}. The collection method in VAMS is similar to that of DBS in that the samples are collected by a finger or heel stick, and the blood is collected by touching the surface of the whole blood droplet on the skin to the absorbent piece on the VAMS device. The VAMS sampling method is user-friendly, because samples can be collected in the clinic by a member of clinical staff or at home by the patient or a caregiver. There are validated methods for quantifying antimicrobial drugs by VAMS-based LC-MS/MS methods, which have been developed, validated, and implemented for analyzing clinical samples^{17, 23}.

Sample Analysis

LC-MS/MS is a common analytical technique that is used for quantifying antibiotics in human matrices, typically in plasma or serum samples¹³. With the advancement in technology, more advanced instruments have been developed that readily detect and quantify very low concentrations of analytes with little sample volume, which make DBS and VAMS suitable for quantitative analysis. Bioanalytical assays require adequate sensitivity for the antibiotics to cover the therapeutic range for clinical application. Therefore, a suitable calibration range needs to be established for accurate and precise quantitation. Optimization of the parameters, column, and selection of the mobile phase of LC-MS/MS is necessary for achieving good sensitivity and reproducibility. A good sensitivity is indicated by a signal-to-noise (S/N) ratio of at least 10 at the desired lowest limit of quantitation (LLOQ). The reproducibility and suitability of a method for a given analysis can be verified by evaluating the resolution, retention time, peak area, and repeatability of a chromatographic system. Robust and optimal extraction conditions with minimal matrix effects are necessary for attaining good assay sensitivity with VAMS or DBS.

DBS Assays

Assay development

The development of DBS assays includes initial experiments for establishing linearity, and preliminary evaluation of analyte recovery and stability, which are necessary for developing a robust assay. The volume of the blood spot is an essential parameter that should be assessed as it may affect analytical data^{16, 18}. Evaluation of blood spot volumes (30 – 50 μL) should be considered if the disks are not punched out from the blood spot. This is especially important when samples are applied to the DBS paper with a capillary pipette. For most DBS assays, a small punch hole (3 – 8 mm in diameter) is removed from the area of the dried spot of whole blood. However, some assays use the entire spot for sample analysis. The next step involves establishing a suitable drying time and extraction conditions for DBS samples containing the analytes at the desired concentration range. Bacterial growth can occur if the samples are not completely dry, which can affect the results of sample analysis¹⁸. The total time for sample drying depends on the size of the spot of whole blood on the DBS paper. DBS cards with the capacity to absorb larger volumes of blood (50 μL) take a longer time to dry. The drying times for the antibiotics discussed in this review vary from 1 to 3 h at ambient temperature (Table 1). Linearity is evaluated for establishing a wide assay range suitable for analyzing clinical samples, which will help reduce the need for re-analysis. Establishing a consistent and reproducible analyte recovery strategy is necessary for ensuring the robustness of an assay. Typically, a combination of aqueous and organic solvents is used for achieving satisfactory recovery. The addition of disodium ethylenediaminetetraacetic acid (EDTA) and deferoxamine mesilate (DFX) to the extraction solvents improves the analyte recovery for rifampicin and clarithromycin²¹. These chelators improve the recovery by sequestering iron(III) and facilitating the precipitation of the dissolved matrix during DBS extraction. Preliminary short-term stability studies for antimicrobials during sample preparation conditions in DBS is useful prior to validation.

Table 1:

Summary of dried blood spot (DBS)-based liquid chromatography with tandem mass spectrometry (LC-MS/MS) assays for determining the concentration of antimicrobial drugs

Drug	Assay range (μg/mL)	Sample collection and processing	Method validation and clinical application
Piperacillin17	3.125 – 200	Sample volume: Pipetted 30 μL on DBS paper; 3.3 – 3.4 μL (3.2 mm disk) Drying time: 1 h at room temperature (RT) Extraction solvent: Water (rehydration); methanol	Inter-assay accuracy: 104.0 – 111.1%. Inter-assay precision: 3.2 – 7.5%. Hematocrit (HCT) effect: Under-estimation at HCT <45% and over-estimation at HCT >45% Recovery: Quality control (QC) low 57%; QC high 66% Matrix effect: QC low 98%; QC high 86% Stability: Not evaluated Clinical application: 1 subject; 2 samples
Tazobactam17	3.125 – 200	Sample volume: Pipette 30 μL on DBS paper; 3.3 – 3.4 μL (3.2 mm disk) Drying time: 1 h at RT Extraction solvent: Water (rehydration); methanol	Inter-assay accuracy: 98.1 – 106.2%. Inter-assay precision: 4.3 – 9.9%. HCT effect: Under-estimation at HCT <45% and over-estimation at HCT >45% Recovery: QC low 59%; QC high 61% Matrix effect: QC low 95%; QC high 90% Stability: Not evaluated Clinical application: 1 subject; 2 samples
Meropenem17	3.125 – 200	Sample volume: Pipette 30 μL on DBS paper; 3.3 – 3.4 μL (3.2 mm disk) Drying Time: 1 h at RT Extraction solvent: Water (rehydration); Methanol	Inter-assay accuracy: 104.0 – 111.1%. Inter-assay precision: 3.2 – 7.5%. HCT effect: Under-estimation at HCT <45% and over-estimation at HCT >45% Recovery: QC low 50%; QC high 43% Matrix effect: QC low 107%; QC high 93% Stability: Not evaluated Clinical application: 3 subjects; 6 samples
Ceftazidime17	3.125 – 200	Sample volume: Pipette 30 μL on DBS paper; 3.3 – 3.4 μL (3.2 mm disk) Drying time: 1 h at RT Extraction solvent: Water (rehydration); methanol	Inter-assay accuracy: 99.5 – 110.1%. Inter-assay precision: 2.3 – 9.9%. HCT effect: Under-estimation at HCT <45% and over-estimation at HCT >45% Recovery: QC low 55%; QC high 55% Matrix effect: QC low 110%; QC high 86% Stability: Not evaluated Clinical application: 3 subjects; 6 samples
Linezolid17	3.125 – 200	Sample volume: Pipette 30 μL on DBS paper; 3.3 – 3.4 μL (3.2 mm disk) Drying Time: 1 h at RT Extraction solvent: Water (rehydration); methanol	Inter-assay accuracy: 97.8 – 106.2%. Inter-assay precision: 2.0 – 8.2%: HCT effect: Under-estimation at HCT <45% and over-estimation at HCT >45% Recovery: QC low 76%; QC high 75% Matrix effect: QC low 106%; QC high 94% Stability: Not evaluated Clinical application: 5 subjects; 10 samples
Linezolid20	0.05 – 40	Sample volume: Pipette 50 μL on DBS paper; 8 mm disk Drying Time: at RT – time not specified Extraction solvent: Acetonitrile	Inter-assay accuracy: 98.7 – 106.3%. Inter-assay precision: 3.5 – 10.2%. HCT effect: Bias <15% for HCT levels 20 – 50% Recovery: QC low 95.5%; QC medium 94.1%; QC high 97.2% Matrix effect: QC low 2.9%; QC medium 8.7%; QC high 1.9% Stability: 1 week at 50°C; 2 months at RT and 37°C Clinical application: 8 subjects
Ceftriaxone19	1 – 200	Sample volume: 6 mm chad Drying time: 1 h at RT Extraction Solvent: 1 g/L of ethylenediaminetetraacetic acid (EDTA) in water (rehydration); 30:70 water-acetonitrile	Inter-assay accuracy: 92.5 – 104.7%. Inter-assay precision: 2.8 – 6.8%. HCT effect: Bias <15% for HCT levels 31 – 67% Recovery: QC low 83.5%; QC medium 83.3%; QC high 84.8% Matrix effect: QC low 103.5%; QC medium 112.6%; QC high 109.2% Stability: 14 h at ambient temperature (35°C); 30 days at 4°C; 21 weeks at −20°C Clinical application: 10 subjects
Rifampicin21	0.15 – 40	Sample volume: Pipette 50 μL on DBS paper; 8 mm disk Drying time: 3 h at ambient temperature Extraction solvent: 1 g/L of EDTA and deferoxamine mesilate (DFX) in water (rehydration); acetonitrile	Inter-assay accuracy: 98.9 – 101.9%. Inter-assay precision: 2.4 – 7.2%. HCT effect: Bias of up to 28% for HCT levels of 20 and 50% Recovery: QC low 70%; QC medium 91%; QC high 87% Matrix effect: DBS: QC low 60%; QC medium 34%; QC high 28% DBS with EDTA: QC low 42%; QC medium 42%; QC high 37% DBS with EDTA and DFX: QC low 102%; QC medium 106%; QC high 96% Stability: 2 months at ambient temperature (25°C); 10 days at 37°C; 3 days at 50°C Clinical application: 12 subjects; 36 samples
Clarithromycin19	0.05 – 10	Sample volume: Pipette 50 μL on DBS paper; 8 mm disk Drying time: 3 h at ambient temperature Extraction solvent: 1 g/L of EDTA and DFX in water (rehydration); acetonitrile	Inter-assay accuracy: 100.2 – 110.5%. Inter-assay precision: 0.0 – 4.8%. HCT effect: Bias <15% at HCT of 20 and 50% Recovery: QC low 99%; QC medium 102%; QC high 95% Matrix effect: DBS: QC low 98%; QC medium 106%; QC high 101% DBS with EDTA: QC low 106%; QC medium 114%; QC high 111% DBS with EDTA and DFX: QC low 100%; QC medium 103%; QC high 102% Stability: 2 months at ambient temperature (25°C); 2 months at 37°C; 15 days at 50°C Clinical application: 12 subjects; 36 samples
Vancomycin26	1 – 100	Sample volume: Pipette 50 μL on DBS paper; 8 mm disk Drying time: 3 h at RT Extraction solvent: 1:1 mixture of water and methanol (v/v) containing 0.1% formic acid	Inter-assay accuracy: 94.4 – 104.6%. Inter-assay precision: 4.7 – 7.6%. HCT effect: Bias <15% at HCT of 25, 40 and 50% Recovery (at 3 HCT levels): QC low 64.2 – 71.9%; QC medium 65.7 – 70.1%; QC high 63.6 – 69.5% Matrix effect: 9.6 – 11.2% Stability: 7 days at 45°C; 14 days at 22°C Clinical application: 29 subjects; 54 samples

Validation

A summary of the validation results of the antimicrobial DBS LC-MS/MS assays is provided in Table 1. Acceptable inter-assay accuracy and precision (±15% of the theoretical value) was achieved for piperacillin¹⁷, tazobactam¹⁷, meropenem¹⁷, ceftazidime¹⁷, linezolid^{17, 20}, ceftriaxone¹⁹, rifampicin²¹, and clarithromycin²¹, by sample quantification with DBS. These DBS antimicrobial assays have been validated and applied to clinical sample analysis. Studies validating the DBS method were assessed according to the guidelines of European Medicines Agency (EMA) and the United States Food and Drug Association for bioanalytical validation.^{16, 24, 25} The accuracy, precision, linearity, dilution integrity, carryover, stability, recovery, matrix effect, and blood to plasma partitioning ratio were evaluated. According to the validation criteria of EMA and FDA, the accuracy and precision of the calibration standards and quality controls (QCs) should not exceed ±15% of the theoretical value (±20% for the lowest limit of quantitation (LLOQ)). The HCT effect is an important factor that should be evaluated in studies validating DBS. HCT impacts the results of sample analysis using DBS because it affects the size of the blood spot on the paper, due to the differences in the viscosity of the blood samples. Typically, a wide range of HCT levels (20% – 60%) is evaluated because patients have varying HCT values depending on the age, gender, and disease state.

Analyte stability in DBS samples

It is essential to assess the stability of drugs in DBS samples, for establishing how the samples should be stored and shipped. With DBS, it is possible to ship the samples at ambient temperatures. While some antibiotics, including linezolid, are stable at extreme conditions (50°C) for up to 1 week, other antibiotics, such as ceftriaxone, may only be stable for 14 h at room temperature^{19, 20}. This information is essential during the transportation of samples from remote areas where cold shipping supplies may not be available. The long-term stability should be assessed at different temperatures (45°C, 22°C, 4°C, −20°C, and −80°C) for determining how long the samples can be stored prior to analysis. The time points of stability studies depend on the intended time of sample storage for batching and analysis. Typically, this is performed at 1 week and 1, 3, and 6 month intervals at the intended storage conditions. This allows the samples to be batched and analyzed together. In routine TDM, the samples are typically analyzed immediately, and back-up samples may be stored for additional analysis.

Clinical application

Understanding the correlation between the plasma and DBS concentrations is essential interpreting the data obtained using DBS and comparing to that of currently established plasma therapeutic ranges. This is achieved by analysis, which involves the collection of venous whole blood, spotting the DBS paper with the whole blood sample, following by centrifugation of an aliquot of the same venous blood sample for plasma collection, which is necessary for comparing the results^19–21. An alternative strategy involves comparing the results from the capillary DBS sample with those of a venous sample collected at the same time¹⁹. As described previously, the sample size necessary for evaluating the correlation between the DBS and plasma samples is typically 40 paired samples collected from at least 40 different patients, which can be used to evaluate the correlation between the two methods¹⁶. The DBS sampling technique has been applied to the bioanalytical assays of several antibiotics for clinical application, as depicted in Table 1, piperacillin, tazobactam, meropenem, ceftazidime, and linezolid have been simultaneously quantified in DBS samples¹⁷. The reproducibility of the DBS strategy was evaluated by reanalyzing a second set of clinical samples. The results differed by more than ±20% in 4 out of 13 cases. The minimal HCT effect was observed in the DBS assay clinical validation study for linezolid with 8 subjects²⁰. The results demonstrated that linezolid had higher concentrations in the whole blood than in the plasma samples and showed a good correlation. A conversion factor of 0.83 was used to calculate the expected plasma concentrations from the concentration of linezolid in the DBS samples. The analysis of ceftriaxone in the DBS samples of 10 subjects showed that the plasma concentration of ceftriaxone and the plasma concentration of ceftriaxone predicted by DBS were in good correlation and showed no statistically significant differences¹⁹. The simultaneous quantification of rifampicin and clarithromycin in 36 samples from 12 subjects showed good correlation between the DBS and plasma samples²¹. The concentrations of vancomycin in capillary DBS and plasma samples collected from 29 patients receiving vancomycin therapy were evaluated in a previous study²⁶. The study demonstrated that the concentrations of vancomycin in the plasma could not be accurately predicted by the DBS strategy, probably due to the variability in the partitioning between the plasma and the red blood cells or the interactions with the substrate of the DBS paper. The comparisons between the DBS and plasma concentrations in these studies were useful in establishing the DBS-plasma correlation, and the DBS method was shown to be promising when corrected for HCT. One of the major limitations of the DBS strategy is that the concentrations of unbound antimicrobial drugs cannot be measured. These studies provide insights into the potential benefits and limitations of DBS for the clinical sample analysis of antimicrobial drugs.

VAMS Assays

Assay development

The initial experiments for developing the VAMS assay involves establishing the linearity, evaluating analyte recovery, and obtaining preliminary stability data from the dried microsample. Dried VAMS makes sample handling, storage, and transportation more feasible²². The variation in the drying times could be an issue with reproducible recovery. Drug extraction is more difficult when a sample becomes more dry. Understanding the effect of drying on VAMS samples is essential for method development and validation. The total time for drying depends on the size of the absorbent tip on the VAMS device. It has been demonstrated that 1 h of drying at room temperature is sufficient for methods using 10 μL VAMS^{17, 23}. The linearity is evaluated for defining a wide dynamic range that includes the expected therapeutic concentration range of antimicrobials. The upper limit of quantitation should be sufficiently high in order to limit the number of samples that require dilution. Consistent analyte recovery is essential for developing a robust VAMS method. Antimicrobial drugs require a rehydration step with 50 μL of water during extraction for enhancing recovery^{17, 23}. Impact-assisted extraction is another strategy for increasing recovery, in which stainless steel beads are used to directly impact the dried blood tip by vigorously shaking at high speeds (vertical strokes per min)²⁷. Small-scale experiments should be conducted for determining the preliminary stability data before initiating validation studies.

Validation

Table 2 provides an overview of the validation results of recently published antimicrobial VAMS-based assays. The inter-assay accuracy and precision of VAMS-based assays for quantifying cefepime²³, piperacillin¹⁷, tazobactam¹⁷, meropenem¹⁷, ceftazidime¹⁷, and linezolid¹⁷ were acceptable. These VAMS assays have been validated and applied to clinical sample analysis. The validation studies were assessed on the basis of the guidelines provided by the EMA and FDA for bioanalytical validation^{22, 24, 25}. The accuracy, precision, linearity, dilution integrity, carryover, stability, recovery, matrix effect, and blood to plasma partitioning ratio were assessed during validation. According to the criteria provided by the EMA and FDA, the accuracy and precision for calibration standards and QCs should not exceed ±15% of the theoretical value (±20% for the LLOQ). Although VAMS eliminates or reduces the HCT effect that is most commonly observed in DBS samples, the HCT effect should be evaluated for VAMS-based assays. Whole blood samples with normal HCT values (41–45%) were used in all the validation experiments. A wide range of HCT levels (20% – 60%) was evaluated because patients have varying HCTs depending on the age, gender, and health.

Table 2:

Summary of volumetric absorptive microsampling (VAMS)-based liquid chromatography with tandem mass spectrometry (LC-MS/MS) assays for determining the concentration of antimicrobial drugs

Drug	Assay range (μg/mL)	Sample collection and processing	Method validation and clinical application
Cefepime23	0.1 – 100	Sample volume: 10 μL Drying time: 1 h at room temperature (RT) Extraction solvent: Water (rehydration); acetonitrile	Inter-assay accuracy: 100 – 108%. Inter-assay precision: 4.27 – 12.2%. Recovery (at 3 hematocrit (HCT) levels): Quality control (QC) low 40.8 – 60.2%; QC medium 43.4 – 57.8%; QC high 48.2 – 62.1% Matrix effect: QC low 96.7%; QC medium 89.5%; QC high 89.8% HCT effect: recovery decreases as HCT increase Stability: 1 week at 4°C; 1 month at −20°C; 3 months at −78°C Clinical application: 1 subject; 7 samples
Piperacillin17	3.125 – 200	Sample volume: 10 μL Drying time: 1 h at RT Extraction solvent: Water (rehydration); methanol	Inter-assay accuracy: 94.9 – 106.0%. Inter-assay precision: 3.2 – 13.6%. Recovery: QC low 86%; QC high 90% Matrix effect: QC low 109%; QC high 107% HCT effect: Bias <15% for HCT levels 30 – 60% Stability: 72 h at RT, 4°C and −20°C; 1 month at −20°C; freeze-thaw acceptable Clinical application: 1 subject; 2 samples
Tazobactam17	3.125 – 200	Sample volume: 10 μL Drying time: 1 h at RT Extraction solvent: Water (rehydration); methanol	Inter-assay accuracy: 92.0 – 115.7%. Inter-assay precision: 6.2 – 9.1%. Recovery: QC low 88%; QC high 83% Matrix effect: QC low 101%; QC high 110% HCT effect: Bias <15% for HCT levels 30 – 60% Stability: 72 h at RT, 4 °C and −20 °C; 1 month at −20°C; freeze-thaw acceptable Clinical application: 1 subject; 2 samples
Meropenem17	3.125 – 200	Sample volume: 10 μL Drying time: 1 h at RT Extraction Solvent: Water (rehydration); methanol	Inter-assay accuracy: 93.5 – 105.3%. Inter-assay precision: 7.1 – 10.6%. Recovery: QC low 86%; QC high 78% Matrix effect: QC low 95%; QC high 109% HCT effect: Bias <15% for HCT levels 30 – 60% Stability: 72 h at RT, 4°C and −20°C; 1 month at −20°C; freeze-thaw acceptable Clinical application: 3 subjects; 6 samples
Ceftazidime17	3.125 – 200	Sample volume: 10 μL Drying time: 1 h at RT Extraction solvent: Water (rehydration); methanol	Inter-assay accuracy: 95.2 – 115.7%. Inter-assay precision: 0.6 – 5.3%. Recovery: QC low 103%; QC high 86% Matrix effect: QC low 104%; QC high 97% HCT effect: Bias <15% for HCT levels 30 – 60% Stability: 72 h at RT, 4°C and −20°C; 1 month at −20°C; freeze-thaw acceptable Clinical application: 3 subjects; 6 samples
Linezolid17	3.125 – 200	Sample volume: 10 μL Drying time: 1 h at RT Extraction solvent: Water (rehydration); methanol	Inter-assay accuracy: 91.6 – 109.3%. Inter-assay precision: 2.3 – 9.5%. Recovery: QC low 94%; QC high 83% Matrix effect: QC low 111%; QC high 95% HCT effect: Bias <15% for HCT levels 30 – 60% Stability: 72 h at RT, 4°C and −20°C; 1 month at −20°C; freeze-thaw acceptable Clinical application: 5 subjects; 10 samples

Stability of analytes in VAMS samples

Information on the stability of drugs in the dried whole blood VAMS samples is necessary for understanding how samples should be handled and stored. For certain drugs, including piperacillin tazobactam, meropenem, ceftazidime, and linezolid, the VAMS samples can be shipped at ambient temperature¹⁷. This makes it convenient to ship samples from remote places where ice or dry ice may not be available. However, for drugs such as cefepime, the samples cannot be shipped at ambient temperature owing to drug instability, which requires shipping under cold conditions. The short-term stability and long-term stability of the drugs should be evaluated at several temperatures (room temperature, 4°C, −20°C, and −78°C). Similar to DBS, the duration of stability depends on the length of time for which the samples have been stored before batching analysis. The stability of analytes in VAMS is typically assessed at 1 week, and at 1, 3, and 6 months, under the intended storage conditions. It is essential to use desiccants when storing samples for stability studies, so as to ensure that the samples remain dehydrated throughout storage. With long-term stability, the samples can be batched for the ease of processing and analysis. Immediate sample analysis may be required for patients undergoing routine TDM, and duplicate samples may be stored for additional analysis, if necessary. The freeze-thaw stability of the analytes may also be evaluated.

Clinical application

VAMS sampling has been applied to clinical studies for quantifying the antimicrobials enlisted in Table 2. The VAMS method was applied for quantifying cefepime in 7 clinical samples obtained from 1 pediatric subject²³. The results demonstrated that the range and sensitivity of the assay were suitable for this VAMS-based LC-MS/MS assay. Piperacillin, tazobactam, meropenem, ceftazidime, and linezolid were simultaneously analyzed in 11 samples obtained from 7 pediatric patients¹⁷. The plasma samples were concurrently collected with the VAMS samples, and the drug concentrations were quantified. However, owing to the limited number of paired samples, the VAMS-plasma correlation could not be established. Another limitation of VAMS is that, in similarity to DBS, the concentration of the unbound antimicrobial drug cannot be measured, which, however, is often required for PK/PD analysis. VAMS is used in clinical research, however, further clinical validation is necessary before it can be applied for routine TDM.

The following conditions should be considered while developing an optimal assay: 1) length of drying time, 2) volume of the VAMS device for best chromatography response, 3) amount of time required for extraction (vortexing, sonication, and centrifugation), and 4) the extraction solvent used. The VAMS validation criteria should involve the requirements put forth in the guidelines of the FDA or EMA for validating bioanalytical methods. Additionally, researchers should consider analyzing the HCT effect and comparing the in vitro drug concentrations measured in the VAMS, whole blood, and plasma samples. This is crucial when comparing the data previously reported in the plasma samples to the data obtained using VAMS technology. As previously described for the DBS samples, the sample size that is necessary for evaluating the correlation between the VAMS and plasma samples is typically 40 paired samples obtained from at least 40 different patients¹⁶.

Gap Analysis

A comparison of the available microsampling techniques may aid in selecting the appropriate procedure for TDM. In a recent study, four antibiotics were simultaneously quantified in DBS and VAMS samples and compared¹⁷. The accuracy and precision of both the matrices were within the acceptable range (± 15%). However, some issues were observed with the DBS samples when the concentration of the drugs were evaluated at extreme levels of HCT. The concentrations of the drugs were underestimated when the levels of HCT were <45%, and the drug concentrations were overestimated when the levels of HCT were >45%, on the basis of the calibration curves prepared using normal levels of HCT (45%). However, this issue was eliminated when VAMS devices were used, which showed acceptable bias (<15%) over a wide range of values of HCT (30–60%). VAMS devices have proven to have minimal HCT effects owing to their ability to collect accurate and precise volumes of whole blood samples.

Plasma and serum are the commonly used matrices for quantifying drugs in TDM. However, drugs with different blood to plasma partitioning profiles, which generally use plasma or serum levels for reference, require new reference ranges based on the values obtained by DBS or VAMS. Therefore, reporting the concentration of the drug by DBS or VAMS would be less informative to a clinician if there is no established reference range for the drug. Clinical validation with the simultaneous collection of VAMS or DBS and plasma samples is necessary for converting the concentrations predicted by DBS or VAMS to the plasma concentrations.

Outlook

Microsampling approaches are useful tools for pediatric research and TDM. These patient-centric forms of sample collection cater to the patient and the clinical team responsible for patient care. TDM in children is more feasible with microsampling, allowing for the collection of samples at multiple time points as only small volumes of blood are required. DBS and VAMS are essential sampling techniques that aid the widespread use of TDM, which will aid the goals of TDM, including the determination of safe and effective drug concentrations, especially for pediatric patients in intensive care units. The at-home sampling capabilities of microsampling techniques, such as VAMS, allow for convenient sample collection at home and direct shipment to the laboratory for analysis. VAMS allows the collection of minute volumes of blood, which benefits patients by limiting the collection of excess blood. Small volumes of blood are sufficient for clinical assays because modern instruments are highly sensitive and can detect low concentrations of drugs and metabolites. With DBS and VAMS, samples can be collected at multiple time points without exceeding the safe limits of blood collection. The collection of microsamples at home ensures flexibility of blood collection, allowing for sample collection in a comfortable place.

Blood microsampling has several benefits, including the collection of small volumes of blood, reducing the risks of infection, alleviating anxiety during venipuncture blood collection, and eliminating the need for a visit to the clinics. However, most drugs require the establishment of a new therapeutic window/target in VAMS for these assays to be implemented for TDM. Another limitation of dried whole blood microsamples is that the concentration of unbound antimicrobial drugs cannot be measured. The level of antibacterial activity is determined from the concentration of unbound drug²⁸. The plasma protein binding property of antimicrobials is an important characteristic because it can impact the PK and PD of the antibiotic²⁹. Further technological advances in microsampling for measuring the plasma and unbound drug concentrations will aid the TDM of antimicrobial drugs in pediatric patients.

Finally, clinical laboratories need to balance the advantages of microsampling techniques in TDM with the challenges of implementation, including assay development and validation, determination of the free drug fraction in the sample, and awareness regarding sampling, when making decisions about whether methods for measuring the concentrations of drug in microsamples should be developed. These decisions should be guided by how microsampling in TDM can improve patient care compared to that of traditional TDM. The decisions should also account for the population(s) in which TDM will be performed, along with the frequency of drug concentration monitoring in patients receiving the drug. Microsampling will perhaps be of greatest benefit for drugs that require repeated monitoring (multiple sampling in a short period of time) or precise monitoring at specific intervals, or in patients in whom traditional blood sampling techniques are difficult, such as infants and children.