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Journal of Pharmaceutical and Biomedical Analysis 147 (2018) 288–296

Contents lists available at ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

j o ur nal ho me page: www.elsev ier .com/ lo cate / jpba

olumetric absorptive microsampling: Current advances andpplications

iranda G.M. Kok, Marianne Fillet ∗

aboratory for the Analysis of Medicines, Centre for Interdisciplinary Research on Medicines (CIRM), University of Liège, Quartier Hopital, Avenueippocrate 15, 4000, Liège, Belgium

r t i c l e i n f o

rticle history:eceived 7 May 2017eceived in revised form 10 July 2017ccepted 26 July 2017vailable online 27 July 2017

eywords:olumetric absorptive microsampling

a b s t r a c t

Recently, volumetric absorptive microsampling (VAMS) has been introduced for the sampling of biolog-ical fluids, and more particularly whole blood, on a porous hydrophilic tip. VAMS enables the collectionof small, accurate and precise blood volumes (10 or 20 �L) regardless of the hematocrit. After drying, thesamples can be stored or directly analyzed. The stability of various compounds in dried samples supportedon VAMS tips varies from one day to a few months at room temperature, and increases at lower temper-atures. The complete tip is used during a simple and straightforward sample preparation. Compoundscan be extracted with a variety of solvents, and thereafter directly analyzed. A design of experiments is

ample preparationtabilityematocritpplications

recommended to determine the optimal extraction conditions for a reproducible recovery. The recoveryof compounds might be influenced by the hematocrit.

In the last two years, various pharmacokinetic and therapeutic drug monitoring studies have beenconducted with VAMS. This review covers the general aspects related with the use of VAMS and itsapplicability is demonstrated through examples.

© 2017 Elsevier B.V. All rights reserved.

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2882. Sample collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2893. Sample preparation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2904. Effect of hematocrit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2905. Sample storage and stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2926. Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2947. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295

Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295

. Introduction

blood spot (DBS) approach, was introduced in 1963 for the screen-ing of metabolic disorders in newborns [1]. Microsampling of wholeblood provides advantages over conventional plasma sampling. Forexample, the collection process is simplified, thereby enabling at-

In the last decades, there has been a growing interest in the

evelopment and use of simple microsampling techniques foruantitative bioanalysis. The collection of small volumes of wholelood on filter paper and subsequent analysis, the so-called dried

Abbreviations: AUC, area under the curve; Cmax, maximum concentration; CD,yclodextrins; DBS, dried blood spot; LC, liquid chromatography; MS, mass spec-rometry; VAMS, volumetric absorptive microsampling.∗ Corresponding author.

E-mail address: marianne.fillet@ulg.ac.be (M. Fillet).

ttp://dx.doi.org/10.1016/j.jpba.2017.07.029731-7085/© 2017 Elsevier B.V. All rights reserved.

home sampling. In addition, dried samples may be shipped andstored at room temperature instead of frozen, and may be clas-sified as non-hazardous. This results in a tremendous reductionin costs. Furthermore, significantly reduced blood volumes (frommore than 200 �L to less than 20 �L) lead to a reduction in thenumber of animals required in pharmacokinetic and toxicokinetic

studies [2].

However, a number of issues and challenges have been associ-ated with the use of the DBS approach, which may affect the qualityof the obtained results. Main issues are the effect of the hemat-

tical and Biomedical Analysis 147 (2018) 288–296 289

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spspartbhib(ritpo2wbs

Fig. 2. Gravimetric determination of the influence of the hematocrit on the volumeof blood absorbed by volumetric absorptive microsampling (VAMS) tips. Six blood

M.G.M. Kok, M. Fillet / Journal of Pharmaceu

crit (volume percentage of blood cells in whole blood) and theomogeneity of the sample. The hematocrit determines the viscos-

ty of the blood and thereby it affects the diffusion of spotted bloodn the filter paper. Most commonly, an accurate sub-punch withinhe dried spot is taken for further analysis. Differences in hemat-crit between samples result in the sampling of inconstant bloodolumes and hence an assay bias. Moreover, non-homogeneity ofnalytes across the blood spot has been reported, which also affecthe accurate quantification of the compounds of interest if a sub-unch is used. Furthermore, the hematocrit and non-homogeneityf the blood spot may have an impact on the extraction of com-ounds, and consequently, on the recovery and matrix effects [3,4].

Various approaches have been proposed to (partly) overcomehe issues with hematocrit and homogeneity of the sample, includ-ng whole spot analysis, pre-cut DBS, correction based on potassiumoncentration and the application of a precise and accurate vol-me on the filter paper [3–6]. Moreover, novel collection devicesave been developed and introduced, including volumetric absorp-ive microsampling (VAMS) [7–9]. VAMS involves the volumetricbsorption and subsequent drying of a liquid sample onto a porousip. It is designed to offer the same advantages as DBS, whereast is supposed to circumvent the problems with hematocrit andomogeneity [7].

This review starts with a description and discussion on generalspects of the VAMS approach, including design, sample collection,ample preparation and the stability upon storage. Characteris-ics of studies involving VAMS [10–31] as well as stability datare summarized in tables. Furthermore, special attention is paido the effect of hematocrit on the quantification of compounds. Inhe second part, the applicability of VAMS is illustrated by givingxamples of pharmacokinetic studies and therapeutic drug moni-oring as well as examples of studies comparing VAMS with otherampling techniques is provided [14,29,32,33].

. Sample collection

A VAMS device consists of an absorbent white tip attachedo a plastic sample handler. The porous hydrophilic tip has beenesigned to absorb a fixed volume of sample by wicking. The vol-me of the sample is controlled by the properties and the amountf the polymeric substrate. VAMS devices are available to collect 10r 20 �L, and can be stored in cartridges (2 samplers), clamshells2–4 samplers) or 96-racks [2,7].

The sample collection procedure involves dipping the leadingurface of the tip into blood at an angle of 45◦. The tip of the sam-ler becomes completely red upon filling with blood within a feweconds [2,7]. Besides blood, VAMS has also been used to collectlasma, urine and saliva [23]. Human whole blood can be takenfter a simple finger or heel prick [10,12,19,22,29,33]. Mouse orat blood can be obtained from the tail vein [14,20,25,30]. Theips should not be immersed in the blood past the tip shoulder,ecause this can result in trapping of excess blood on the plasticandler. Problems with sampling were encountered in the follow-

ng toxicokinetic study of paracetamol in rats [14]. In this study, autterfly infusion set was used to collect blood from the tails of ratssee Fig. 1A). But the blood flow from the tail was faster than theate at which the blood was absorbed by the VAMS tip, resultingn retaining of blood on the handler. The authors suggested thathe extensive blood flow might be caused by the administration ofaracetamol [14]. The sampling method was adapted in the coursef the study in order to better control the bleeding rate using a

3G needle to fill a luer lock with blood (Fig. 1B). The needle hubas removed from the tail and vertically placed in a polystyrene

lock, after which a sample was taken using VAMS [14]. Thiry et al.howed recently that this sampling procedure can be simplified by

samples were weighed at six different laboratories and the respective blood volumeswere calculated based on the density of the samples. The average calculated volumesare depicted. Reprinted with permission from Ref. [2].

generating a drop of blood at the tail with a 23G needle, after whichthe blood can be directly taken with a VAMS device (Fig. 1C) [30].

A cross-laboratory study showed that the VAMS technologyenables the collection of an accurate and precise blood volumeregardless of the hematocrit [2]. Samples of human and rat blood(10 �L) with different hematocrit values (20, 45 and 65%) weretaken and the weight of the absorbed blood was determined (n = 6for each hematocrit value from 6 laboratories). In addition, theblood density was assessed by weighing exact volumes of 10 �Ltaken with a pipette (n = 6 for each hematocrit value). The volumeabsorbed with the VAMS devices was calculated by dividing theweight of the absorbed blood by the mean density of the blood.This resulted in an overall average volume of 10.6 ± 0.4 �L (stan-dard error of the mean), collected within the hematocrit range(Fig. 2). The overall variability across the different laboratories andthe hematocrit values was only 8.7%. However, there was a largedifference between the minimum (9.1 �L) and maximum (13.1 �L)absorbed blood volume. Furthermore, the absorbed volumes in twolaboratories were for each blood sample above average, indicat-ing a potential systematic bias (Fig. 2). This might be explained bythe different weighing procedure to determine the densities of theblood samples in these two laboratories. The blood samples wereadded to a vial containing blood with the same hematocrit value,and the increase in weight was assessed. Using this approach, asmall amount of blood may remain in the pipette tips, leading toan underestimation of the density, and hence, an overestimationof the blood volume. In the other laboratories, blood samples wereremoved from a vial and the decrease in weight was determined,thereby avoiding this potential bias [2]. In another study, it wasshown that the abuse of the tips during sampling can also lead tovariability in the amount of blood retained on the VAMS tips. Forinstance, handling the tip with greasy fingers led to a higher amountof retained blood (9% more). In contrast, a highly variable reductionwas observed when the VAMS devices were handled with poorlydried hands. Abusing the tips after sampling (e.g. tips dropped ortouched against various other materials) led to a loss of sample ofup to 19% [7].

So, in general VAMS can be used to take samples directly fromhumans and animals after a simple prick in the finger or tail, respec-tively. VAMS allows the collection of precise and accurate volumes

independent of hematocrit level. However, care should be taken toavoid any excess blood on the sample handlers and the abuse ofthe tips leading to different sample volumes and analyte concen-trations.

290 M.G.M. Kok, M. Fillet / Journal of Pharmaceutical and Biomedical Analysis 147 (2018) 288–296

Fig. 1. Various sampling strategies using volumetric absorptive microsampling (VAMS) devices. The use of a butterfly infusion set (A1) resulted in retaining excess bloodo trollet needlp

3

fsrifnr[Vvwdt

iiombdTV[bamhoatoptc

n the sample handler due to a high blood flow (A2). The bleeding rate can be conaken (B2). Simplified sampling with VAMS by generating a blood drop with a 23Germission from Ref. [14,30].

. Sample preparation

After collection of the blood, the samples are most often driedor at least two hours at room temperature (Table 1). Thereafter,amples can be shipped and/or stored before the analysis is car-ied out. During the sample preparation, the whole sampling tips used for the extraction of the compounds of interest. There-ore, issues of non-homogeneity experienced by DBS samples areegated with the VAMS technique. In some studies the tip wasemoved from the holder before the extraction was performed11,13–15,17–19,21–23,29,31], whereas in others the completeAMS device was used [10,12,16,20,24–28,30]. Tip removal pre-ents the extraction of any excess blood on the sample handler,hereas automation of processes is possible when the completeevice is used, because of its design (same dimensions as pipetteip) [7].

The selection of the extraction solvent and other conditionss of utmost importance. Ideally, the extraction procedure resultsn a highly reproducible and maximum recovery of the analytesf interest and a low recovery of other compounds to minimizeatrix effects. The optimization of the extraction conditions can

e performed via an experimental design [34]. The optimal con-itions are really dependent on the analytes. As can be seen inable 1, the extraction solvent in the performed studies withAMS ranged from 100% water [10–12,17,29] to 100% organic

14,18,20,23–26,28,30,31]. Moreover, in some studies an acid orase was added to enhance the recovery [12,13,16,17,24,27,31]. Inddition, the extraction time varies between studies, from a fewinutes to one hour. Increasing the extraction time may result in a

igher recovery of the analytes. However, a difference in recoveryf only 3% was observed for fosfomycin between mixing times of 30nd 60 min. Still, the recovery was maximum 63.9% [26]. In addi-ion, an increase in extraction time can also maximise the extractionf potential interfering compounds, which may lead to signal sup-

ression [16]. An internal standard is at the earliest be added tohe extraction solvent [13–16,20–22,24–26,29–31]. So, it might notompletely compensate for analyte losses.

d by using a 23G needle to fill a luer lock (B1), after which a blood sample can bee (C1) and directly approaching the drop with a VAMS device (C2). Reprinted with

Overall, VAMS offers advantages to other sampling techniquesin terms of sample preparation. The sample pretreatment is rel-atively simple, since there is no need for centrifugation beforeanalyte extraction. Furthermore, the complete tip is processed,thereby avoiding a sub-punch step, which is commonly used forDBS samples. Therefore, potential cross-contamination of samplesis eliminated. The disadvantage is that the VAMS sample can only beprocessed once. So, multiple samples should be taken beforehandif repeated analyses are indicated.

4. Effect of hematocrit

As described earlier, VAMS enables the accurate collectionof blood volumes across a broad hematocrit range [2,7]. How-ever, various studies report an effect of hematocrit level onthe accuracy of the quantitation of the compounds of inter-est [13,14,18,21,24,26,31]. For instance, Parker et al. observedan inverse correlation between accuracy and hematocrit for theanalysis of fosfomycin [26]. A positive bias was observed at lowhematocrit values, whereas a negative bias was found for bloodsamples with a high hematocrit. Potential causes of this bias arechanges in recovery of the analyte or matrix effect across thehematocrit range. Since no signal suppression or enhancementfor fosfomycin was found, the inverse correlation was causedby changes in recovery. Indeed, the recovery of fosfomycin fromthe VAMS samples differed across the tested hematocrit values.A recovery of 45.6–48.9% was observed at a hematocrit of 52%,whereas the recovery of fosfomycin was 60.4–63.9% for blood sam-ples with a hematocrit of 27%. This difference could not be causedby variability in the absorbed volume on the VAMS tips, becausean exact volume was spotted on the tips using a pipette [26]. Asimilar trend of a higher recovery at lower hematocrit is observedin six other studies [13,14,18,21,24,31], whereas no differences in

extraction yield and matrix effect were found when cannaboidswere quantified in blood samples with hematocrit values rangingfrom 30 to 60% [28]. The lower recovery at higher hematocrit levelshas been explained by the larger amount of erythrocytes entrapping

M.G

.M.

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291

Table 1Overview of sample preparation conditions for samples taken with volumetric absorptive microsampling (VAMS).

Compound Sample type Dry (h) Extraction solvent Extraction conditions Additional sample preparation Ref.

Fe Human blood 2 milli-Q water vortex for 5 min acid digestion overnight at 110 ◦C [10]Various metals Human blood 2 milli-Q water vortex for 2 min acidification and addition of IS [11]Various metals Human blood 4 1% nitric acid in ultrasonic bath for 1h – [12]Caffeine and paraxanthine Human blood 2 methanol-water (8:2, v/v) +

0.01% formic acid + ISin a thermomixer at 1000 rpm and22 ◦C

centrifugation at RT (10 min, 10000 × g),supernatant diluted with 0.01% formic acid.

[13]

Paracetamol Rat blood 2 methanol + IS on a lateral shaker for 1h 9-fold dilution with methanol-water (1:4, v/v) [14]Steroids Rat blood 3 methanol-water (1:1, v/v) + IS ultrasonication for 5 min precipitation with zinc sulfate, centrifugation

(10 min, 23000 × g)[15]

Hepcidin Human blood 2 methanol-water-formic acid(75:25:0.1, v/v/v) + IS

soak for 5 min, followed by agitation ina thermomixer at 500 rpm and 20 ◦C

centrifugation (10 min, 3400 × g), evaporationof supernatant under vacuum andreconstitution inacetonitrile-water-trifluoroacetic acid(33.8:66.2:0.1 v/v/v)

[16]

Sulfur mustard-albumin adducts Human plasma overnight 50 mM ammoniumbicarbonate

sonication for 15 min and intensevortex mixing

addition of pronase solution and incubation for4 h at 37 ◦C, addition of acetonitrile andcentrifugation (5 min at 10270 × g), drying ofsupernatant and reconstitution in 0.5% formicacid + IS

[17]

Miltefosine Human blood 3 methanol mixing for 15 min at 1250 rpm andvortex for 30 s

20-fold dilution of highly concentratedsamples with methanol, addition of IS

[18]

5-methyltetrahydrofolic acid Human blood 2.5 0.1% Triton X-100 in 200 mMMES containing 20 g/L ascorbicacid and 1 g/L DTT (pH 5)

vortex for 2 s and centrifugation for10 min at 15400 × g

polyglutamate deconjugation [19]

Various drugs Rat blood 2 acetonitrile-methanol (1:1,v/v) + IS

shaking for 1 h at RT 3-fold dilution with water [20]

E6005 Human blood overnight acetonitrile-water (1:1, v/v) +IS

sonication for 15 min and vortex for10 s

– [21]

Hydroxyurea Human blood 2–3 acetonitrile-water (8:2, v/v) + IS shaking for 15 min at 40 ◦C centrifugation (20 min, 14000 × g), evaporationof supernatant with nitrogen stream,reconstitution in 98% acetonitrile

[22]

Cathinone analogues Human blood, urine and oral fluid 1 methanol ultrasound agitation for 15 min andvortex for 1 min

evaporation under vacuum and reconstitutionin acetonitrile-water (1:1, v/v) containing 0.1%formic acid

[23]

Emixustat Human blood 2 methanol containing 1%ammonium hydroxide and IS

sonication for 15 min and vortex for15 min

evaporation at 40 ◦C with nitrogen,reconstitution in methanol-water (3:7, v/v)containing 0.1% formic acid

[24]

Estetrol Mouse blood 2 methanol + IS incubation for 5 min, agitation for5 min with vortex mixer

phospholipid removal with Ostro® -plate,evaporation under vacuum at 50 ◦C for 1 h,reconstitution in methanol-water (1:1, v/v)

[25]

Fosfomycin Human blood 2 methanol + IS on a lateral shaker for 30 min at1200 rpm

– [26]

Vancomycin Human plasma 0.5–56 acetonitrile-water (1:1,v/v) + 0.1% formic acid

on a lateral shaker for 30 min at1200 rpm and in an ultrasonic bath for10 min

centrifugation for 5 min at 4000 × g [27]

Cannabinoids Human blood 1 methanol vortex for 1 min and ultrasoundagitation for 15 min

evaporation with nitrogen stream,reconstitution in acetonitrile-water (1:1, v/v)containing 0.1% formic acid

[28]

Hydroxychlorquine Human blood 3 water + IS vortex for 30 min protein precipitation with 70% perchloric acid,centrifugation for 5 min at 14000 rpm

[29]

Itraconazole and metabolite Rat blood 2 acetonitrile-methanol (6:4,v/v) + IS

incubation for 5 min, agitation for5 min with vortex mixer

phospholipid removal with Ostro® -plate,evaporation under vacuum at 50 ◦C for 1 h,reconstitution in methanol-water (1:1, v/v)

[30]

Various drugs Human blood 2 methanol-acetonitrile (1:1,v/v) + 1% formic acid + IS

vortex for 1 h centrifugation at 3000 rpm for 15 min [31]

DTT: dithiothreitol, IS: internal standard, MES: 2-(N-morpholino)ethanesulfonic acid hydrate, RT: room temperature.

292 M.G.M. Kok, M. Fillet / Journal of Pharmaceutical a

Fig. 3. The effect of the hematocrit level at the accuracy of the quantitation of theinvestigational drug E6005 (circles) and its metabolite M11 (squares) at low (black)and high (white) concentrations. Whole blood samples were absorbed on volumetricabsorptive microsampling (VAMS) devices and extracted (A) without or (B) with as

tesaw

ooamwtat1castocaaibmwoaf

showed a decrease in signals of 29–35%. The stability of emixustatin the VAMS samples was improved at lower storage temperatures.

onication step of 15 min. Reprinted with permission from Ref. [21].

he analyte in the pores of the VAMS tip, thereby complicating itsxtraction [18,26]. Remarkably, Kip and coworkers found an oppo-ite trend when blood samples with various hematocrit values werenalysed with DBS compared to VAMS. The recovery of miltefosineas higher from DBS samples with a high hematocrit [18].

In one study, it has been shown that the effect of the hemat-crit on the recovery can be (partly) overcome with the inclusionf a sonication step during the extraction procedure [21]. The bio-nalysis of the novel phosphodiesterase 4 inhibitor E6005 and itsetabolite M11 was executed with VAMS devices. Blood samplesith hematocrit levels of 20.0, 43.9 and 66.4% were used to study

he impact of the hematocrit on the recovery of the two analytest low and high concentrations (3 and 160 ng/mL). At low hema-ocrit, the extraction yield with acetonitrile-water (1:1, v/v) was00%, whereas at a high hematocrit level the recovery for the low-oncentrated samples was decreased to 69.9 and 73.4% for E6005nd M11, respectively. The decrease for the high-concentratedamples was less, but still significant (80.4 and 83.6%). In ordero achieve a hematocrit-independent recovery, a sonication stepf 15 min was included in the extraction. This resulted in signifi-ant higher recoveries. Still, there was a trend of lower recoveriest higher hematocrit levels. For instance, the recoveries for E6005nd M11 at 3 ng/mL increased to 83.4 and 89.0, respectively. Thempact of the hematocrit was also apparent in the accuracy of theioanalytical method. Without sonication, a positive bias of maxi-um 31.7% was observed for the samples with a hematocrit of 20%,hereas a maximum negative bias of 25.3% was found for a hemat-

crit of 66.4% (Fig. 3A). The addition of a sonication step resulted in

n average positive bias up to 16.7%. Still, there is a trend presentor a hematocrit-dependent bias (Fig. 3B) [21].

nd Biomedical Analysis 147 (2018) 288–296

Moreover, the hematocrit effect on the recovery can be dimin-ished by careful selection of the extraction solvent. The influenceof the hematocrit on the extraction of the drugs metoprolol, mida-zolam, atorvastatin, tamoxifen and amiodarone varied when usingdifferent extraction solvents [31]. For example, using methanol adecrease in recovery of over 20% was observed for metoprolol, ator-vastatin and tamoxifen as blood hematocrit increased from 20 to70%. The recoveries appeared to be more consistent with methanol-acetonitrile (1:1, v/v) as extraction solvent. Indeed, in this solventmixture, the recoveries for atorvastatin and midazolam were sim-ilar in the hematocrit range of 20–70%. For the other three drugsa decrease of maximum 15% was observed at 70% hematocrit. Themore consistent recoveries might be a result of an effective desorp-tion of the compounds from the VAMS tips due to the good solvationability of methanol and the strong elution strength of acetonitrile[31].

Although VAMS enables hematocrit-independent sampling ofaccurate blood volumes, differences in hematocrit may lead tochanges in recovery of the analytes of interest. However, it hasbeen shown that this negative effect might be (partially) resolvedby careful selection of the extraction solvent and by the inclusionof a sonication step during sample preparation.

5. Sample storage and stability

VAMS enables at-home and remote sampling of whole bloodvia a finger prick. Thereafter, samples might be shipped andstored at room temperature before analysis provided that thestability of compounds in whole blood supported on the VAMStips is guaranteed. Numerous studies have been conducted inwhich the stability of various compounds was tested (Table 2)[10,13,14,16–19,21–26,28–30]. The stability was studied underdifferent storage conditions, such as temperatures ranging from−80 ◦C to +50 ◦C and time frames of 1 up to 120 days. In manystudies, the difference in signals for the respective compounds wasless than 15% between the first and last day of the stability study.Therefore, the compounds were considered stable in dried wholeblood for the studied period [10,13,14,17–19,21,23,28,29]. How-ever, depending on the molecules, a trend towards a decrease instability can be observed. For instance, Kip and colleagues found adecrease in signal over time when miltefosine was stored on VAMStips at room temperature and at 37 ◦C. Despite the decline, the sta-bility was still acceptable at the end of the 30 days study period[18]. Remarkably, there were no stability issues observed by thesame research group when blood spiked with miltefosine was col-lected via DBS and stored for 162 days at temperatures ranging from−70 ◦C to 37 ◦C [35]. It should be noted that the storage conditionsdiffered slightly, since VAMS samples were stored in the dark inclamshell storage containers and DBS samples in sealed aluminumbags with three desiccant packages [18,35].

A clear decrease in signal over time was observed for emixu-stat, estetrol, fosfomycin, hepcidin, hydroxyurea and itraconazole[16,22,24–26,30]. Miao et al. tested the stability of emixustat whichis an investigational drug for eye diseases. Emixustat-spiked bloodsamples absorbed on VAMS tips (low and high concentration) wereleft at ambient temperature, stored refrigerated (4 ◦C) or frozen(–20 ◦C) for up to three months before extraction was carried out.The samples stored at ambient temperature were stable for oneweek with a mean bias (n = 6) of maximum 12%. Samples pro-cessed after three months of storage under the same conditions

Refrigerated samples were stable for one month, whereas stabilityof frozen samples was demonstrated for three months.

M.G.M. Kok, M. Fillet / Journal of Pharmaceutical and Biomedical Analysis 147 (2018) 288–296 293

Table 2Stability of compounds in dried whole blood supported on VAMS tips.

Compound Temp. (◦C) Stability (days) Remarks Ref.

Fe −20 4* Storage in closed tips holders [10]4 4*

21 28*

60 4*

Caffeine and paraxanthine RT 82* Clamshells and stored in zip-closure plastic bags withdessicant

[13]

60 4*

Paracetamol −20 2* Stored in zip lock plastic bags with silica gel [14]AT 7*

40 2*

Hepcidin RT 6 Test period: 24 days [16]Sulfur mustard-albumin adducts 21 9* Stability tested with different relative humidity [17]

30 9*

Miltefosine 22 30* Stored in clamshell storage containers in the dark [18]37 30*

5-methyltetrahydrofolic acid −20 17* Stored in resealable bags with dessicant [19]E6005 RT 2* [21]Hydroxyurea −80 60* 4 ◦C: after 30 days 25% signal decrease for a concentration

of 0.5 �g/mL and 52% signal decrease at 60 �g/mL[22]

−20 60*

4 <30Synthetic cathinones RT 7* Stored in the dark in sealable bags with dessicants [23]Emixustat −20 92* Test period: 92 days [24]

4 30AT 7

Estetrol RT <2 Test period: 42 days [25]Stored in the darkAfter 2 days 40% signal decrease

Fosfomycin −20 120* RT: after 75 days 31.4% and after 120 days 53.1% signaldecrease

[26]

RT 10 50 ◦C: after 4 days 78.4% signal decrease50 <4

Cannabinoids RT 30* Stored in the dark in sealable bags with dessicants [28]Hydroxychloroquine 22 10* [29]

50 1*

Itraconazole and metabolite RT 1 Test period: 38 days, stored in the dark [30]After 35 days 52.4% (itraconazole) and 58.6% (metabolite)signal decrease

R sampb

wiutat

aadumi

patwodppf−tu

T: room temperature, AT: ambient temperature, *Stability of compounds in driede stable for a longer period.

Moreover, it has been shown that the stability of compounds inhole blood supported on VAMS tips can be extended by extract-

ng the compounds directly and storing the dried extracts at −80 ◦Cpon further analysis. Using this approach, blood samples con-aining itraconazole and estetrol could be stored for at least twond six weeks respectively, instead of less than two days at roomemperature for non-extracted samples [25,30].

Parker et al. studied the stability of the antibiotic fosfomycint room temperature and −20 ◦C for a period of four months. Inddition, the stability at 50 ◦C for four days was examined [26]. Aifference in peak area of +1.6%, −31.4% and −53.1% was observedpon storage at room temperature for ten days, two and a halfonths and four months, respectively. Storage at 50 ◦C resulted

n a decrease in peak area of 78.4%.Application of fosfomycin-containing whole blood onto DBS

aper and subsequent storage at room temperature resulted in lower decrease in peak area, namely a decline of 14.0% afterwo and a half months. However, when plasma with fosfomycinas applied to the same type of DBS paper, the decrease was

nly 4.9% after a storage period of three months. So the observedegradation of fosfomycin in dried whole blood samples might beartly induced by blood components which are removed during thereparation of plasma samples. Interestingly, peak areas for fos-

omycin were on average 15.2% higher after four months storage at20 ◦C. This might be due to changes in extraction recovery across

ime [26]. This potential recovery aspect has been further exploredsing plasma samples containing vancomycin which is known to

les on VAMS tips was only tested for the indicated time period. Compounds might

be stable [27]. The recovery was assessed by comparing the resultsof two different samples analysed with liquid chromatography(LC) coupled to tandem mass spectrometry (MS). Exactly 10 �L ofvancomycin-spiked plasma (8 and 80 �g/mL) was pipetted on aVAMS tip and the tip was placed in the extraction solvent of 50%acetonitrile containing 0.1% formic acid. For the other sample, drug-free plasma was applied on a VAMS tip and added to the extractionsolvent spiked with vancomycin. Before addition to the extractionsolvent, the samples were kept in the dark at room temperaturefor various time periods. The recovery for vancomycin decreasedfrom 101 to 87% (8 �g/mL) and from 102 to 97% (80 �g/mL) whenincreasing the drying time from 2 to 56 h. A more significant effectof drying time on recovery was observed when the same experi-ment was carried out with DBS paper. The recovery of vancomycinwas 59 and 60% (2 h) and 32 and 41% (56 h) at 8 and 80 �g/mL,respectively [27]. So, the results of this study indicate that concen-tration results may be unreliable if there are important differencesin storage time between samples and/or calibration standards.

In general, many compounds appeared to be stable during thecomplete tested period. If the stability of compounds is guaran-teed at room temperature, costs for shipping and storage can besignificantly reduced since there is no need to freeze the samples.However, the stability of some compounds could not be assured for

a long time at room temperature. The stability can be increased bystoring the samples at lower temperatures before or after analyteextraction.

2 tical and Biomedical Analysis 147 (2018) 288–296

6

tcsOssocutshmmprwf4s1rwtjb

apcTprsicwCcttCtiv

bCscfoobaDctotrnw

Fig. 4. Comparison of caffeine (a) and paraxanthine (b) concentrations obtainedwith three different sampling methods, namely volumetric absorptive microsam-pling (VAMS), dried blood spot (DBS) and whole blood in EDTA tubes. Differencesin concentrations (%) between the conventional approach (plasma EDTA tubes) and

94 M.G.M. Kok, M. Fillet / Journal of Pharmaceu

. Applications

Different methods with VAMS have been developed and some-imes validated to analyse compounds with various physiochemi-al properties. Proof-of-concept studies have demonstrated the fea-ibility of VAMS for various applications [13,15,17,19,22,23,25,28].ne proof-of-concept study involved the collection of plasma,

aliva and urine instead of whole blood for the determination ofynthetic illicit drugs [23]. Plasma was obtained by centrifugationf blood collected in EDTA-tubes. Urine and saliva samples wereollected in glass tubes and subjected to centrifugation for ten min-tes (4000 rpm, at 4 ◦C). An internal standard solution was addedo the supernatants. These samples were directly processed (wetample) and after VAMS sampling followed by a drying step of oneour at room temperature. The synthetic drugs were extracted withethanol by means of ultrasound agitation (15 min) and vortexixing (1 min). The extracts were vacuum dried and reconstituted

rior to LC–MS/MS analysis. Samples were taken from five self-eported drug users. The concentrations of the drugs obtainedith VAMS were in good agreement with the amounts of drugs

ound in the wet samples. For instance, plasma concentrations of-methylethcathinone were 49 ng/mL (VAMS) and 53 ng/mL (wetample) for one of the drug users. The urine concentrations were26 ng/mL and 125 ng/mL when analysed as VAMS and wet sample,espectively. The concentration of 4-methylethcathinone in salivaas below the limit of quantification. Unfortunately, the different

ested matrices could not be concurrently collected for each sub-ect. Therefore, plasma concentrations obtained using VAMS cannote related to urine and saliva concentrations [23].

A few of the validated methods involving VAMS have really beenpplied in more extensive studies to determine pharmacokineticroperties of the respective compounds and to assess the appli-ability of VAMS for therapeutic drug monitoring [14,29,32,33].he first application of VAMS was a toxicokinetic study in ratserformed by Denniff et al. [14]. In this study, two groups of fiveats each were orally dosed with paracetamol (600 mg/kg/day) foreven days. Blood samples were drawn from the tail vein at var-ous time points after dosing (see Section 2). VAMS was used toollect three samples at each time point for one group of rats,hereas for the other group blood was collected in EDTA tubes.oncentration-time profiles were constructed to determine toxi-okinetic parameters, including the area under the curve (AUC) andhe maximum concentration (Cmax). The mean AUC was similar forhe two groups (20% within each other). However, the obtainedmax was up to 40% higher for the VAMS derived profiles comparedo profiles obtained after blood collection in EDTA tubes. Consider-ng the small group size, this difference can be caused by biologicalariability [14].

De Kesel and coworkers compared DBS, VAMS and venous wholelood collected in EDTA tubes leading to plasma samples [13].oncentrations of caffeine and paraxanthine were determined inamples originating from 96 hospital patients. Differences in con-entrations were observed between the three sampling techniquesor both compounds (Fig. 4). Concentrations of both compoundsbtained with DBS analysis were markedly affected by hemat-crit. However, it has been shown before that concentrations cane effectively corrected for this hematocrit bias by means of anlgorithm based on potassium concentrations [5]. Furthermore,BS concentrations were over the whole hematocrit range lowerompared to concentrations obtained with plasma samples. In con-rast, VAMS concentrations were significantly higher irrespectivef hematocrit. Differences between VAMS and plasma concentra-

ions were on average 12.3 and 15.4% for caffeine and paraxanthine,espectively [13]. This is a similar bias as observed in the toxicoki-etic study of paracetamol in rats. However, it should be noted thathole blood concentrations after VAMS sampling are compared

VAMS (white spots) or DBS (black spots) are plotted against hematocrit values ofthe respective blood samples. The broken lines represent linear regression lines.Reprinted with permission from Ref. [13].

to plasma concentrations. The correlation between whole bloodand plasma concentrations is dependent on the hematocrit and theextent of protein binding. Therefore, results might not be compareddirectly, and the conversion or correlation of the concentrationsshould be investigated systematically.

Thiry et al. assessed the in vitro release and bioavailability ofitraconazole in four different pharmaceutical formulations, namelyone commercially available product (Sporanox

®) and three for-

mulations, which were made via hot melt extrusion [32]. Theformulations were prepared with Soluplus

®as the main poly-

mer matrix with or without the addition of a super-disintegrant(AcDiSol

®) and cyclodextrins (CD). Six rats successively received

the formulations with a washout period of one week. VAMS deviceswere used to collect fourteen blood samples from each rat withina 48 h time frame. Itraconazole and its main metabolite hydroxy-itraconazole were extracted from the VAMS devices using a simpleextraction procedure. A validated LC–MS/MS method allowed theconstruction and comparison of pharmacokinetic profiles of theseextracted compounds of interest (Fig. 5) [30,32]. Whole bloodconcentrations of both compounds were significantly higher afteradministration of the three in-house produced formulations com-pared to concentrations obtained with Sporanox

®. Calculated AUCs

and Cmax values were also significantly higher for the formulationsproduced by hot melt extrusion. Furthermore, these in vivo resultscould be partly related to in vitro dissolution rates of the variousformulations [32]. The true advantage of VAMS in this study wasthe reduction of the number of rats required to test the various for-

mulations. Fourteen samples could be collected per rat without theneed to sacrifice the animals. Therefore, all formulations could beexamined using the same animals, thereby also suppressing inter-individual variability.

M.G.M. Kok, M. Fillet / Journal of Pharmaceutical an

Fig. 5. Pharmacokinetic profiles of itraconazole (ITZ) and its metabolite hydroxy-itraconazole (OH-ITZ) after oral administration of four different formulations(containing 10 mg ITZ) to rats. Volumetric absorptive microsampling was used tocollect samples at fourteen different time points. The average concentrations forebR

aaiwptlobarVlrs

hVpaccAsamacmra

[

[

[

ach time point ± standard deviations (n = 6) are plotted. Significant differencesetween the profiles were observed with p-values below 0.01 (**) and 0.001 (***).eprinted with permission from Ref. [32].

VAMS has also been used to collect human blood samples for thenalysis of HbA1c and hydroxychloroquine [29,33]. Verougstraetend coworkers tested the feasibility of VAMS and DBS for the mon-toring of HbA1c in diabetes patients. The VAMS and DBS samples

ere collected at home and analysed after an average storageeriod of three days. In addition, blood and VAMS samples wereaken during consultation and immediately processed and ana-ysed. There was a poor agreement between the concentrationsbtained after sampling at home (both VAMS and DBS) and thelood samples collected upon consultation. For this reason, theuthors conclude that home-sampled VAMS and DBS are not aeliable tool for the monitoring of HbA1c. However, processing theAMS samples directly (without drying) resulted in similar HbA1c

evels compared to standard blood samples [33]. Further research isequired to investigate this difference, particularly concerning thetability of HbA1c upon storage.

Qu et al. applied VAMS for therapeutic drug monitoring ofydroxychloroquine on 54 patients with rheumatoid arthritis [29].AMS samples were collected after a finger prick with a lancetuncture. In addition, venous blood was collected in EDTA-tubesfter which a protein precipitation and centrifugation step wasarried out. After sample pretreatment, concentrations of hydroxy-hloroquine and three metabolites were assessed with LC–MS/MS.verage concentrations of hydroxychloroquine were 990.1 (±92.3tandard error of the mean) and 873.4 (±83.4) ng/mL for the VAMSnd venous blood samples, respectively. Despite this difference inean concentration, Deming regression analysis showed a good

greement (slope of 1.08) between VAMS and venous blood con-entrations. Similar trends were observed for the three quantified

etabolites of hydroxychloroquine with regression slopes in the

ange of 1.04–1.11. So in this study, VAMS appeared to be a valuablepproach for therapeutic drug monitoring at home [29].

[

d Biomedical Analysis 147 (2018) 288–296 295

7. Concluding remarks

This review focused on the use of VAMS for quantitative bioanal-ysis. Numerous studies showed that precise and accurate volumesof whole blood, independent of hematocrit, could be sampled usingVAMS devices. Sample preparation is relatively straightforward,although the extraction solvent appears to be the critical parameterto optimize. Furthermore, on the contrary to DBS samples, the com-plete VAMS tip is used for the analysis, thereby avoiding the needfor sub-punching. Numerous validated quantitative methods havebeen reported and these methods have been successively appliedfor therapeutic drug monitoring and the determination of phar-macokinetic parameters. Despite these advantages, a few issuesstill need to be addressed before widespread application of VAMSis foreseen. Not all compounds are stable enough in dried bloodsupported on the VAMS tips. Moreover, a hematocrit-dependentrecovery was apparent for some compounds. Finally, discrepanciesin concentrations obtained after VAMS and other sampling tech-niques have been observed for particular compounds, indicatingthat the correlation between whole blood and plasma concentra-tions should be investigated systematically.

Acknowledgement

The authors acknowledge the University of Liège and theEuropean Union (EU) for financial support in the context of theFP7-PEOPLE-COFUND-BeIPD project.

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