dried blood spots: concepts, present status, and future perspectives in bioanalysis

ReviewDrug Testing

and Analysis

Received: 16 January 2014 Revised: 22 February 2014 Accepted: 24 February 2014 Published online in Wiley Online Library

(www.drugtestinganalysis.com) DOI 10.1002/dta.1646

Dried blood spots: Concepts, present status,and future perspectives in bioanalysisAbhisheak Sharma,a,b Swati Jaiswal,a,b Mahendra Shuklaa,b

and Jawahar Lala,b*

Over the past several years, dried blood spot (DBS) sampling technique has emerged as a pertinent method in both qualitativeand quantitative bioanalysis context. In the DBS method, the blood sample is directly soaked on to a paper (with or withouttreatment). After drying it can be analyzed by modern analytical, immunological, or genomic detection systems. Severaladvantages of the DBS technique such as low blood volume requirement, transportation and storage without special treat-ment, better analytes stability, enhanced clinical cooperation in clinical trials, and reduced unforeseeable exposure of analyststo biohazards, make it the most appropriate blood sampling technique. This review illustrates the information available onthe DBS method which may serve as a single window for investigators in the field of bioanalysis. Also, it explores theproficiency and appliance of the DBS method in pharmacokinetic (PK), therapeutic drug monitoring (TDM), toxicokinetic(TK), metabolomic, and disease diagnosis. Copyright © 2014 John Wiley & Sons, Ltd.

Keywords: dried blood spot (DBS); pharmacokinetic (PK); therapeutic drug monitoring (TDM); biomarker; bioanalysis

* Correspondence to: Jawahar Lal, Pharmacokinetics & Metabolism Division,CSIR-Central Drug Research Institute, Jankipuram Extension, Sitapur Road,Lucknow - 226031, India. E-mail: [email protected]

a Pharmacokinetics & Metabolism Division, CSIR-Central Drug Research Institute,Lucknow - 226031, India

b Academy of Scientific and Innovative Research, New Delhi, India

Introduction

Implementation and effectuation of dried blood spot (DBS)technology in the realm of therapeutics and health sciences hassimplified the conventional blood collection and analysis process.DBS is anticipated to be a promising surrogate of established liquidbio-matrices (plasma/serum) for pharmacokinetic (PK) andtoxicokinetic (TK) studies and could even surpass them. Ivar Chris-tian Bang, the father of Modern Clinical Microchemistry, pioneeredthe DBSmethod for quantification of blood sugar.[1] After half a cen-tury of Bang’s DBS application, [1] Guthrie and Susi [2] in 1963,reported the DBS method for the analysis of phenylketonuria in ne-onates and thereafter the DBS method gained popularity inbioanalysis. Blood is the most preferred and regulatory acknowl-edged biological sampling matrix for in vivo concentration assess-ment in variety of species. [3,4] Venipuncture, the traditional bloodsampling technique, has a major drawback of being an invasiveand painful technique. In clinical trials, venipuncture may beresponsible for poor volunteer recruitment. Moreover, serialsampling is difficult in small animals during PK and TK studies dueto the requirement of large blood volume withdrawal. Unlike thedownsides of venipuncture, blood sampling through DBS isminimally invasive (finger prick versus venipuncture in humans;blood collection through tail vein versus retro-ocular or intra-cardiacin laboratory animals). Following the collection of blood drops on apaper, drying, and transportation, the blood spot is extracted andanalyzed in the laboratory. DBS cards are considered to be lessbiohazardous in comparison to plasma samples in view of a factthat blood is in dried form; all the proteins, pathogens, and enzymesare inactivated on the card and bacterial growth is prevented. Theless invasive nature of DBS and the use of micro-volume bloodsamples have made it a very useful sampling method for neonataland juvenile subjects. DBS reduces the number of steps precedingdrug analysis (i.e. requirement of cold chain during transportationand storage as well as centrifugation) (Figure 1). By using the DBS

Drug Test. Analysis (2014)

technique, public health laboratories screened more than 95% ofall newborns in the USA for inborn metabolic disorders. [5] For PKand TK studies in small animals, such as rats or mice, the DBSmethod can facilitate rich sampling and these samples are stableat a wide range of temperatures. This technique has been reportedto be suitable for drugs which are susceptible to photodegradation.Forced and natural photodegradation experimental data has shownthat nifedipine and omeprazole exhibit higher photostability whenspotted and stored on various DBS paper than that in water, plasma,or whole blood. [6] DBS-assisted analysis has extensive applicationsin PK and TK studies, therapeutic drug monitoring (TDM), diseasescreenings, testing of doping substances and metabolomic studies.It is being widely utilized in the pharmaceutical industries, hospitalsand research centers, particularly where blood or plasma samplevolumes are low, difficult to collect, store, process or transport.

Tools and techniques

Analysis of component(s) (exogenous and/or endogenous) in bio-logical matrix by the DBS samplingmethod involve following steps:

Selection of paper

DBS cards are composed of non-cellulose or cellulose (filter paper)matrix of specific pore size and thickness. Nowadays, variouscommercial DBS cards are available, namely Whatman 903 cards,

Copyright © 2014 John Wiley & Sons, Ltd.

Figure 1. Comparison of DBS method (A) with conventional blood sampling techniques (B) Abbreviations: * = optional step, ψ = represents the stepsfor limitation of conventional venipuncture over DBS

A. Sharma et al.

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FTA DMPK type-A, B, C cards and FTA Elute cards (GE Healthcare,Piscataway, NJ, USA), as per the type of analytical requirements.Routinely, Whatman 903 cards are basically used in newbornsscreening, FTA DMPK type A, B, C cards are used in PK/TK studiesand FTA Elute cards are intended mainly for collection and purifi-cation of DNA for downstream analysis. All types of DMPK cardsare available in two forms: regular and indicating. Indicating cardsare useful for colourless samples like urine, plasma, synovial fluid,and cerebrospinal fluid. DMPK type A and B cards are chemicallytreated with proprietary reagents that, on contact cause lyses ofcells, denature proteins, inactivate enzymes, and prevent thegrowth of bacteria. These coated cards are prepared to cause lysesof both cellular and nuclear membranes to expose nucleic acidswith good stability for storage and analysis. [7] These DMPK cardsalso inhibit the enzymatic degradation of several analytes namelyprocaine and acetyl salicylic acid from esterases which are presentin the blood. These enzymes are denatured and inactivated whenblood is spotted on the card leading to enhanced analyte stability.[8] DMPK-C and Ahlstrom 226 cards (ID Biological Systems, Green-ville, SC) are not treated with any chemical; therefore, there are noimpregnated chemicals to interfere with the analysis. Moreover,proteins will not be denatured thus DMPK-C and Ahlstrom 226cards may be better choice for protein based biomolecules analysis.US Food and Drug Administration (FDA) has approved three DBScards, namely Ahlstrom 226-K062932,Whatman 903 and PerkinElmer226 under 21 CFR 862.1675 asmedical device for blood specimen col-lection. [9] Non-cellulose DBS cards (Bond Elut DMS Card, AgilentTechnologies, Santa Clara, CA, USA) are also commercially availablefor DMPK research. They are claimed to be superior in form of im-provedmass spectrometry (MS) signal, less effort in punching and he-matocrit independent spot homogeneity. [10] In-house treatment ofcards has also been reported for compound specific stability. Guowenet al. [11] used citric acid solution on a DBS card to stabilize their drugcandidate (KAI-9803) from thiol-disulfide exchange. Irrespective of

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unknown blood volume spotting on conventional DBS cards, Gabrielet al. [12] developed a disposable chip. It has self-actuated dissolvablevalves which meter and transfer exact volume of the blood inmicrolitres.

Specimen collection

For TDM and diagnosis of diseases in humans, whole bloodsample is collected from a finger, toe, or heel prick with the aidof sterile disposable lancet or surgical blade. For PK and TKstudies in rat and mouse, blood can be collected from the caudalvein. For qualitative purpose, the DBS specimen can be preparedby cautiously applying a few freshly drawn blood drops from afinger/toe prick on a card. When the objective is quantitativeanalysis, a measured amount of blood volume is gently appliedon a DBS card with the aid of a pipette or capillary tube. In suchcases, use of an anticoagulant is a perquisite for appropriatespotting. Although heparin or ethylenediaminetetraacetic acid(EDTA) can be used as an anticoagulant, the latter is moresuitable. However, EDTA may cause interference in MS but it hasadvantages over heparin in its mixing and drying abilities alongwith calcium-dependent phospholipases and ester hydrolases inhi-bition. [13–15] Use of anticoagulants significantly affects the results intelomere length measurements in quantitative polymerase chainreaction (qPCR) based analysis in DBS. [16]

Volume of blood to be spiked on the DBS card depends on thesensitivity of bioanalytical method or instrumentation facility. Thepipette tip should be held just above the card allowing dropformation and soaking onto the surface. The pipette tip shouldnot be touched repeatedly to the surface of the card as itmay damage the paper leading to sample inhomogeneity.Multi-layered DBS spot formation may occur due to repeatedapplication of blood drops in the same collection circle. All theseshould be avoided as they make the specimen invalid and lead to

14 John Wiley & Sons, Ltd. Drug Test. Analysis (2014)

An overview on dried blood spots

Drug Testing

and Analysis

misinterpretation of results. Improperly labelled cards as well asdark, supersaturated, clotted, discoloured, contaminated, andserum rings containing spots are considered as invalid specimens.As described by Ren et al., [17] autoradiography may be a solutionfor visualization of uneven distribution of analyte(s) on DBS spots.Hematocrit value, which represents the measure of packed cell vol-ume in blood, affects the size of DBS and is usually the main causeof the bias in results. Blood samples with different hematocrit valuepossess variable viscosity and spreadability on the card resulting indissimilar spot size. In such situations, conventional assumption ofdirect proportionality relationship between sample volume andspot size fails. Hence, fixed spot area measurement cannot beapplied for quantitative purposes. Also, there are a few other factorssuch as the type of card material and the environment of spottingwhich contribute to the uneven distribution of analyte on DBScards. Several approaches were tried to overcome the effects ofthese factors. [18] Blood samples can be spotted for a pre-cut driedblood spots (PCDBS); this will nullify the effect of the hematocrit byextracting the whole spot without punching a specific area. [19] Useof capillaries with fixed volume dispenser for finger pricking anddevelopment of novel cards with less propensity to impact of he-matocrit and drying milieu, may be implemented. [18] Capiau et al.[20] reported a method for determining the hematocrit value inany DBS by measuring potassium in the DBS extract using aroutine clinical chemistry analyzer. This add-on technique canassess the haematocrit-dependent intra-individual variability andfurther facilitate non-volumetric DBS sampling.

Drying of the card

After sample collection, the DBS cards are generally driedhorizontally for 2–3 h on a card rack at room temperature orunder nitrogen flow and controlled humidity. [21] The timerequired for drying the card will depend on the type of card aswell as the sample volume. Care should be taken not to lay ahand on wet spots. Cards should not be stacked or allowed totouch other surfaces while drying. Exposure of the DBS specimento any milieu like direct sunlight, dust, or flying insects should beavoided as that may compromise its integrity. In most of theresearch papers, analytes were reported to be stable after dryingand in between analysis tenure. However, drying of the card isthe crucial step for unstable analytes. Various modifications likechanges in pH, [11] temperature, and humidity are recommended.[22] Heat stabilization can also be a solution for metabolicallyunstable drugs. According to Blessborn et al., [23] enzymaticactivity of blood can be reduced by drying DBS card at 95 °C for30 s. They found that oseltamivir, cefotaxime, and ribavirin are sta-ble after heat stabilization but artemether and dihydroartemisininwere unstable due to their decomposition at 60 °C. StabilizerTM

instrument from Denetor (Gothenburg, Sweden) is commerciallyavailable for heat stabilization of DBS cards. After drying, thesecards are suitable for analysis, transportation, or storage.

Storage and transportation

In contrast to conventional biological matrices, DBS provides ahuge simplification in the arena of storage and transportation.Unlike plasma/serum samples, DBS requires neither freezers forstorage nor bulky volumes of dry ice for shipping which are costlyand need special care. Barring the humidity factor, which has sig-nificant influence on specimen stability and elevates the chancesof bacterial growth, DBS cards can be shipped and stored at

Drug Test. Analysis (2014) Copyright © 2014 John Wiley

ambient temperature. For protection from environmental humid-ity, DBS cards are advised to be carefully wrapped and packed insealable heavy duty plastic bags with adequate desiccant and ahumidity indicator to find out at what time the desiccant has tobe replaced. DBS cards are considered as non-regulated andexempt material as per US Department of Transportation (DOT)and the US postal service. Properly labelled DBS cards packets,which clearly convey the biohazardous nature of the contentinside package to transportation personnel and other employees,can be shipped to analytical laboratories through mail, courier, orexpress mail delivery services. For establishing sample integrityand safety from occupational exposure of hazardous bloodsamples, basic triple packaging technology is used for DBS cardshipment. Triple package comprises of primary container, sec-ondary container, and a third covering of high quality paperenvelope with an affixed or printed version of the internationalbiohazard symbol. DBS packages can be stored at cool and dryplace as such or can also be kept in polystyrene foam boxes untiltransportation to laboratories. If long-term stability of certainanalytes at room temperature is not established on DBS cards,the packed DBS cards with desiccant can be stored in laboratoryfreezers until analysis to minimize analyte degradation. [24–27]

Addition of internal standard (IS)

Addition of IS in the DBS method is a tricky task. In most of the cases,IS is added before extraction, either directly in extraction tube or inextraction solvent. In such circumstances, IS does not provide the ex-traction efficiency from the DBS spot and act as an external stan-dard. [28] In a second method, IS can be pre-spotted on DBS cardbefore addition of blood sample for better results. [29] IS can alsobe sprayed on a DBS card for homogeneity across the spotensuring reproducibility. [28] Spraying or spotting of IS on aDBS card is a worthwhile method compared to the addition ofIS in extraction tubes or extraction solvent, as the latter is incapa-ble of finding extraction variability of analyte(s) from the DBS.

Extraction and analysis

Dried cards can be punched out with various available diameterpunching tools (manual, semi-automated, and automated).Punched dried cards can be used directly (by microfluidics) orby extraction of analytes with suitable extraction solvent. Extrac-tion solvent should be optimized as per the solubility profile ofthe analyte(s) with consideration of minimizing extraction ofinterfering endogenous impurities. Extraction efficiency from fixedDBS can be improved by addition of liquid ammonium. [30] After ex-traction, samples are subjected to analysis. Liquid chromatography-tandem mass spectrometry (LC-MS/MS), desorption electrosprayionization mass spectrometry (DESI-MS), gas chromatography–mass spectrometry (GC-MS), matrix assisted laser desorption massspectrometry (MALDI-MS), MALDI time-of-flight mass spectrometry(MALDI-TOF-MS), high performance liquid chromatography (HPLC),isoelectric focusing (IEF)-HPLC, direct laser desorption (LD) TOF-MS,inductively coupled plasma mass spectrometry (ICP-MS), laserablation (LA) ICP TOF-MS, polymerase chain reaction (PCR), enzymelinked immunosorbent assay (ELISA) and microfluidic chip have suc-cessfully been coupledwith theDBSmethod for qualitative andquan-titative analyses of blood samples. [13] An extraction-free direct sprayionization technique is reported byManicke et al. [31] in which the an-alyte is converted into gas phase ions using solvent electrosprayby applying a high voltage (3500 V) to the wet substrate.

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A. Sharma et al.

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Automation

Commercial instruments are available for fully automated onlineDBS sampling and analysis. Online automated tools (ABS2; InstechSolomon, Plymouth Meeting, PA, USA and Culex; BASi, WestLafayette, IN, USA) are capable of collecting blood from freelymoving laboratory animals and can be coupled for serial sampling(in microlitre of blood volume) on DBS cards with high throughputand accuracy. Automated Sample Card and Prep (SCAP) system(Prolab, Reinach, Switzerland) can be coupled with LC-MS/MS foronline drug analysis. Ganz et al. [32] applied this system for the on-line analysis of bosentan and its three metabolites in human DBSwith adequate accuracy and precision. Deglon et al. [33] haveinnovated an online analytical method in which sample can beanalyzed by online desorption of DBS spot in an inox cell bycolumn switching mode with assurance of purification andseparation of samples. This online method paves a way to highthroughput by reducing various steps of bioanalysis likecentrifugation, extraction, vortexing, drying, or reconstitution.

Calculation and correlation with plasma/serum results

Blood level of drug is the sum of levels of drug in red blood cells(RBCs) and plasma. Often due to differential binding of drug tospecific blood components, concentration in plasma and RBCsvaries; hence considering whole blood concentration as equiva-lent to plasma concentration becomes inappropriate. Beforecomparing the plasma/serum levels with DBS levels, parameterslike hematocrit (H) and RBC to plasma partitioning (KRBC/plasma)or blood-plasma partitioning (KBlood/plasma) should be consideredas DBS results may differ from serum or plasma results. Hemato-crit is calculated by dividing packed RBC volume by total bloodvolume and packed RBC volume can be determined simply bycentrifugation of heparinized blood in microhematocrit tubes at10 000 rpm for 5 mim. A conventional method for estimatingKRBC/plasma or KBlood/plasma involves spiking of drug in plasma/plasma water containing suspended RBCs, followed by equilibra-tion, centrifugation and then quantification of drug concentra-tions in plasma/plasma water and RBCs. [34] Once theseparameters are known, plasma concentration can be calculatedfrom DBS results by using the following equation.

Calculated plasma concentration ¼ Analyte concentration in DBS

1-Hð Þ þ H*KRBC=plasma

h i

(1)

If KRBC/plasma or KBlood/plasma is:

1. greater than one, DBS will have higher drug levels than plasmaor serum results and DBS will have better PK/PD correlation.

2. equal to one, DBS results should be identical to plasma orserum results and DBS can be an alternate for plasma study.

3. less than one, DBS results should have lower analyte levels thanplasma or serum. In this condition, the DPS (dry plasma spot)method can be applied with two layered polymer film whichseparates plasma from whole blood without centrifugation. [35]

DBS is the best suitable method for sampling of drugs havinghigher affinity to RBC in comparison to plasma.


DBS applications

Application of DBS technology has gained momentum in variousfields including neonatal metabolic screening, TDM, preclinical andclinical PK, TK, forensic, biological, and immunological sciences.Literature reports on the applications of DBS in the investigationsof drug, biomarkers or diseases are compiled in Tables 1 and 2.

Preclinical pharmacokinetic/toxicokinetic study

Serial blood micro-sampling is possible with DBS in rodentswithout changing their haemodynamic balance. By implementingthe DBS method, animal requirements can be reduced to theextent of at least 60%. Preclinical PK studies in rodents requiresampling of blood with 1–2mL per sample at multiple time-points. Therefore, the studies are done by sparse sampling. Thus,the low sampling volumes required for DBS also facilitates serialsampling from the same animal, offering improved data qualityover composite sampling paradigms frommultiple study animals.Additionally, it will reduce the cost of experiment by reducing thenumber of animals especially in the case of transgenic orknockout model studies. Also, it eliminates the harvesting stepof serum/plasma and reduces the quantity of new chemicalentities (NCE) required during drug discovery and developmentprogramme. Dainty et al. [36] have compared the DBS method withwhole blood results and reported that the results from both aresimilar in relation to drug concentration-time profile, area undercurve (AUC), stability, precision, variability and data accuracy. Forthe compoundswith high RBC to plasma partition coefficient, usingplasma as an analytical matrix leads to significant errors in pharma-cokinetic-pharmacodynamic (PK-PD) calculation due to haemolysisof blood. These errors can be overcome by replacing plasma withDBS. [37] Liang et al. [38] have successfully applied the DBS methodfor discovery PK study of multiple NCE by cassette dosing.

Therapeutic drug monitoring

TDM is the quantification of drug(s) concentration in patients’blood with time to optimize the dose regimen. It helps theclinician as well as the patient regarding drug safety, as doseoptimization can be achieved without compromising the efficacyof therapeutically potent molecules. TDM may help to minimizethe incidences of drug resistance as antibiotic or antiviral drugconcentration levels can be maintained within the therapeuticwindow. As shown in Table 1, several methods are reported forTDM using DBS as a sampling technique. The DBS method isespecially suitable for TDM in neonates. It can also be used forestablishing TDM units in developing countries to save the initialinvestment as well as processing cost of test samples.

Population disease control and clinical pharmacokineticstudy

Multi-centric clinical investigations can be studied easily with thehelp of the DBS technique. Congenital or genetic disorders in ne-onates are being investigated by the DBS method. Many diseasescan be screened by using the DBS sampling technique (Table 2).The toe or finger prick is always better than venipuncture as itcan improve volunteer recruitment in clinical studies. DBS is al-ways advantageous for multiple sampling in remote areas wheretransportation, cold chain facilities, or intravenous blood sam-pling may reduce the efficiency of mass policy implementation.


Table 1. Applications of DBS for investigation of drugs

S. No. Drug name Application Analytical technique Reference

1. Acetaminophen TK studies LC-MS/MS [64]

2. Actinomycin-D PK studies LC-MS/MS [65]

3. Amodiaquine, chloroquine

and chlorthalidone

Preclinical PK studies LC-MS/MS [66]

4. Amprenavir TDM and PK studies LC-MS [67]

5. Antiepileptic drugs (AEDs) [levetiracetam,

lamotrigine, phenobarbital,

carbamazepine and

its active metabolite

carbamazepine-10,11 epoxide]

TDM HPLC [68]

6. Atazanavir TDM and PK studies LC-MS [67]

7. Atenolol TDM and PK studies LC-TOF-HRMS [69]

8. Bisoprolol, ramipril and simvastatin TDM LC-HRMS [70]

9. Bosentan PK studies LC-MS/MS [32]

10. Busulfan TDM LC-MS/MS [71]

11. Caffeine PK studies LC-MS/MS [72]

12. Canrenone Neonatal screening LC-MS [73]

13. Centchroman and 7-demethylated

centchroman

PK and drug-drug interaction study LC-MS/MS [50]

14. Chloroquine and desethylchloroquine PK studies HPLC-UV [74]

15. Choline theophyllinate PK studies Fluoroimmunoassay [75]

16. Clonidine Preclinical and clinical PK studies LC -MS/MS [76]

17. Clozapine TDM HPLC [77]

18. Cocaine Control of subjects suspected of

driving under the influence

of psychotropic substances.

HPLC coupled with

spectrofluorimetric detection

[78]

19. Cyclosporin A TDM Radioimmunoassay

(RIA)/ LC-MS/MS

[79,80]

20. Cyclosporin A and tacrolimus TDM LC-MS/MS [81]

21. Darunavir, etravirine, raltegravir

and ritonavir

TDM LC-MS/MS [82]

22. Darunavir, saquinavir, atazanavir,

amprenavir, lopinavir, ritonavir,

etravirine, efavirenz and nevirapine

TDM and PK studies LC-MS/MS [67]

23. Dasatinib Metabolite profiling LC-MS/MS [83]

24. Dexamethasone Neonatal PK studies LC-MS [84]

25. Dextromethorphan and dextrorphan Enantioselective Method LC-MS/MS [85]

26. Diazepam PK studies LC-MS/MS [86]

27. Donepezil Preclinical PK studies LC-MS/MS [87]

28. Efavirenz TDM RP-HPLC-UV [88]

29. Ertapenem TDM and PK studies LC-MS/MS [89]

30. Estradiol Clinical studies RIA [90]

31. Ethyl glucuronide (EtG) and

ethyl sulfate (EtS)

Diagnosis of recent alcohol uptake LC-MS/MS [91]

32. Etravirine TDM and pharmacological research LC-MS/MS [67,92]

33. Everolimus TDM LC-MS/MS [93]

34. Exenatide Clinical PK studies ELISA [94]

35. Exendin-4 PK and TK studies LC-MS/MS [95]

36. Fluoxetine, norfluoxetine,

reboxetine and paroxetine

TDM, toxicological analysis

and PK studies

NICI-MS-MS [96]

37. Flurbiprofen PK studies LC-MS/MS [72]

38. Gabapentine Preclinical and clinical PK studies LC-MS/MS [97]

39. Gemifloxacin Preclinical PK studies HILIC with

fluorescence detector

[98]

40. Guanfacine Clinical PK studies LC-MS/MS [99]

41. Linezolid TDM LC-MS/MS [100]

42. Lopinavir TDM and PK studies LC-MS [67]

(Continues)


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Table 1. Continued


43. Losartan Preclinical PK studies LC-MS/MS [101]

44. Metformin TDM HPLC-UV [102]

45. Metformin and sitagliptin PK studies LDTD-MS/MS [103]

46. Methadone TDM HPLC [104]

47. Metoprolol Preclinical, clinical PK

and doping studies

LC-MS/MS [105]

48. Metronidazole PK/PD studies in neonatal patients HPLC [106]

49. Midazolam PK studies LC-MS/MS [72]

50. Monodesethylchloroquine, chloroquine,

cycloguanil and proguanil

PK and epidemiological studies HPLC [107]

51. Morphine and 6- acetylmorphine Forensic study LC-MS/MS [108]

52. Moxifloxacin TDM LC-MS/MS [109]

53. Mycophenolic acid TDM and post-marketing

clinical studies

LC-MS/MS [110]

54. Naproxen Bioavailability and PK studies LC-MS/MS [111]

55. Nevirapine and Efavirenz TDM LC-MS/MS [112]

56. Nifedipine Photodegradation experiments LC-MS/MS [6]

57. Non-conjugated testosterone, testosterone

glucuronide (TG), androsterone glucuronide

(AG) and etiocholanolone glucuronide (EtG)

Sports drug testing / dope testing GC-MS [113]

58. Nofovir and emtricitabine PK studies LC-MS/MS [114]

59. Omeprazole PK studies LC-MS/MS [72]

60. Opiates, cocainics and amphetamines Illicit drugs determination

in suspected cases of

driving under the influence

of drugs

LC-MS/MS [115]

61. Oseltamivir Pediatrics TDM and PK studies LC-MS/MS [29]

62. Paclitaxel Preclinical PK studies LC-MS/MS [116]

63. Peginesatide Sports drug testing LC-MS/MS [117]

64. Pioglitazone TK studies LC-MS/MS [118]

65. Posaconazole Clinical PK studies LC-MS/MS [119]

66. Pramipexole Preclinical PK studies LC-MS/MS [120]

67. Propranolol PK studies in neonates or

young infants

LC-MS/MS [121]

68. Quinine and 3-hydroxyquinine Clinical PK studies HPLC with

fluorescence detection

[122]

69. Raltegravir TDM and pharmacological research LC-MS/MS [123]

70. Ramoplanin Clinical PK and bioequivalence studies LC-MS/MS [124]

71. Ranitidine TDM in pediatrics samples LC-MS/MS [125]

72. Rifampicin TDM HPLC-UV [126]

73. Rifaximin TDM, assessing adherence to

medications and preventing

toxicity in a clinical setting

LC-MS [127]

74. Rosiglitazone PK studies LC-MS/MS [72]

75. Rufinamide TDM LC-MS/MS [128]

76. Salmeterol TDM HPLC [129]

77. Sisomicin and netilmicin TDM in geriatric and paediatric patients HPLC with pre-column

derivatization and

fluorimetric detection

[130]

78. Sulfadoxine and pyrimethamine TDM and epidemiological studies HPLC-UV [131]

79. Sulfadoxine and sulfamethoxazole HPLC-UV [132]

80. Tacrolimus TDM LC-MS/MS [133]

81. Tacrolimus and creatinine TDM and assessment of renal function LC-MS/MS [134]

82. Tacrolimus, sirolimus, everolimus and

cyclosporin A

TDM LC-MS/MS [135]

83. Tafenoquine TDM HPLC with fluorescence

detection

[136]

(Continues)

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Table 1. Continued


84. Theophylline TDM, particularly suited for domiciliary care Fluorescence polarization

immunoassay (FPIA)

[137]

85. Topiramate TDM FPIA and LC-MS/MS [138]

86. Vincristine Clinical pharmacological studies LC-MS/MS [65]

87. Zatebradine PK studies LC-MS/MS [139]

88. Zidovudine Monitoring of zidovudine therapy among

HIV-infected pregnant mothers and their new borns

RIA [140]

89. Zopiclone Forensic studies LC-MS/MS [141]


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DNA-based diagnosis of infectious and genetic disorders can beperformed through DBS-coupled PCR tests such as humancytomegalus virus (HCMV) detection, diagnosis of HIV, T-cell re-ceptor excision circle (TREC), Fabry disease mutation and spinalmuscular atrophy (SMA) diagnosis, etc. [13,39–43]

In the USA, the DBS method was evaluated and validated bycentres for disease control and prevention for new born screen-ing tests. The DBS method has also been reported for a plethoraof analytes, biological markers, nutrients, antibodies, and en-zymes for diagnosis and monitoring of epidemiological diseases.[5,44] Snijdewind et al. [45] especially recommended use of the DBSmethod in epidemic surveillance, screening, and follow-up of vi-ral infection and its treatment in remote areas.

Conducting a clinical trial is always a costly affair. In case of aworldwide multi-centric clinical study, cold chain (freezer or dryice) supported transportation of biological samples from the clin-ical facility to the analytical laboratory involves huge capital in-vestment along with stability issues. After drying, spotted DBScan be transported at ambient temperature. The proficiency ofDBS can reduce the overall clinical trial budget by up to 50%. [46]

Other applications

DBS is applicable to most of the blood-related qualitative orquantitative analysis. Doping analysis of banned substances caneasily be performed with the help of DBS technology. Sportsevent grounds or training facilities are not always near an analyt-ical laboratory. By using the DBS sampling technique, blood sam-ples from sports personnel can easily be collected, transported,and analyzed to get unbiased results in a cheap, fast, automated,discrete, and minimal invasive mode. Thomas et al. [47] analyzed26 prohibited compounds in a single analysis using DBS coupledwith LC-MS/MS consisting of a quadrupole mass filter, higher col-lision dissociation (HCD) cell, and an orbitrap detector. DBS is notjust a technique for whole blood analysis; it can also be used foranalysis of banned substances in urine or saliva [48] andimplemented for dope testing. The DBS method is equally appli-cable for plasma or serum analysis in medico-legal and forensicapplications. Kong et al. [49] performed metabolomic profiling of695 detectable and 137 identifiable markers through DBS usingGC-MS and recommended it as a substitute of plasma formetabolomic studies. Lal et al. [50] used the DBS method for PKdrug-drug interaction study of centchroman and its metabolitewith carbamazepine, indicating its applicability in PK drug-druginteraction study. Wijnen et al. [51] applied the DBS method forDNA isolation in pharmacogenetics and found that the DNA iso-lation from the DBS technique is faster, cheaper, and easier thancommercially available DNA isolation kits.


Limitations of DBS technique

Although the DBS technique has gained massive success in thearena of bioanalysis and experienced a surge of interest in severalfields, a plethora of questions exist whose answers are up in theair. The most distinct advantage of the DBS is the use of lowblood volume which is correspondingly a limitation of the DBSmethod as it can only be coupled with highly sensitive analyticaltechniques. It is not as amenable as plasma or serum sampleswith the most common analytical instrument like HPLC coupledwith UV-Visible detector. Also, the DBS sampling technique is in-adequate for air sensitive or volatile analytes. [21,52] Basically, DBStechnology involves use of capillary blood from volunteers inclinical studies; however, there are plausible chances that capil-lary blood analyte concentration may vary from venous blood.In that case, DBS results may differ from those obtained with tra-ditional venous sampling leading to less appropriateness of thistechnique for such analytes. It has been reported that paraceta-mol has significantly higher concentrations in finger-prick bloodthan venous blood. [53,54] For the calculation of bioavailability, se-lection of the bio-matrix is an essential task. In case of low clear-ance and saturable blood cell binding of the compounds, theblood cell to unbound plasma concentration ratio will vary withconcentration. As a result, whole blood or DBS will not be ableto calculate accurate bioavailability as the blood ratios of AUCswill differ. Also, DBS is not a worthy technique for compoundshaving minimal blood cell uptake (KBlood/plasma ≤ 0.55).

[37] DPScan be a solution in such situation. Multi-layer spotting and selec-tion of punching site on DBS card is the general cause of inaccu-rate results in quantitative analysis. This problem can be solvedby using whole blood spot with fixed volume for analysis ratherthan a specific punched part of DBS. There are chances that theanalyte level in certain test samples could cross the calibrationlimit. Then, plasma or serum test samples can be diluted with di-lution integrity testing which is a tricky and tedious task withDBS. Hematocrit value influences the spread of blood on theDBS card and partially influences the results with significant in-ter-subject variability. Apart from hematocrit, spot homogeneityand compound degradation are other problems. The EuropeanBioanalysis Forum (EBF) has expressed concern on changes inextraction recovery of analyte(s) after the aging of the DBS card.In long-term stability studies of DBS cards, concentration varia-tion in relation to storage period has also been reported foramino acids. It is reported that carnitine levels increased by7.6% per year for first five years, followed by a 1.4% decrementper year. [55] Using a non-cellulose DBS card, long-term stabilitystudies assessment, analytical method modification, or addition


Table 2. Applications of DBS for investigation of biomarker or diseases

S. No. Drug/ biomarker/ disease Application Analytical technique Reference

1. 17α- hydroxypregnenolone and

17α-hydroxyprogesteroneNewborn screening for congenital

adrenal hyperplasia (CAH)

LC–MS/MS [142]

2. 17α-hydroxyprogesterone Diagnosis of neonatal CAH Microwave-assisted silylation

(MAS)-GC-MS

[143]

3. 25-hydroxyvitamin D3 Neonatal screening LC-MS/MS [144]

4. Acid beta-D-glucosidase, acid

sphingomyelinase,

chitotriosidase and alpha-N-

acetyl-galactosaminidase

Diagnosis of Gaucher and

Niemann-Pick patients

Enzyme analysis [145]

5. Adenosine-deaminase defect Adenosine and 2-deoxyadenosine testing MS/MS [146]

6. Adrenal steroids Diagnosis of neonatal CAH LC-MS/MS [147]

7. Alloisoleucine Newborn screening for Maple Syrup

Urine Disease (MSUD)

LC-MS/MS [148]

8. Androstenedione Home monitoring of steroid replacement

therapy in congenital adrenal hyperplasia.

Fluorometric immunofunctional

assay

[149]

9. Apolipoprotein A-I Time and temperature of storage of samples

and standards necessary for Apo A-I assays

using ELISA methodology

ELISA [150]

10. Apolipoprotein b Screening programme for new

borns as lipid metabolism

in neonates.

ELISA [151]

11. Babesiagibsoni (asian

genotype) infection

For mass screening of canine

babesiosis in dogs

PCR [152]

12. Biotinidase Lysosomal storage diseases (Pompe, Fabry,

Gaucher, Hurler and Hunter)

Microfluidics [153]

13. C26:0-lysophosphatidylcholine X-linked adrenoleukodystrophy

newborn screening

LC-MS/MS [154]

14. Carnitine and acylcarnitines Clinical diagnostic and public health

newborn screening

LC-MS/MS [155]

15. Cd4+ lymphocytes For Cd4+ lymphocytes counting in

HIV patients

ELISA [156]

16. Cd4+ t-cell CD4+ lymphocyte counting for

the monitoring of antiretroviral treatment

ELISA [157]

17. Ceruloplasmin Newborn screening for Wilson’s disease LC-MS/MS [158]

18. Citrulline Severity screening for enteral disorders LC-MS/MS [159]

19. Cortisol, 4-androstene-3,17-dione

and 17-hydroxyprogesterone

Screening for congenital adrenal hyperplasia LC-MS/MS [160]

20. Cotinine Biomarker of maternal smoking LC-MS/MS [161]

21. Cotinine and trans

3’-hydroxycotinine

Screening to assess second-hand smoke

(SHS) exposure in children

LC-MS/MS [162]

22. C-reactive protein Diagnosis of cardiovascular disease Enzyme immunoassay (EIA) [163]

23. Cytomegalovirus infection Early diagnosis of viral congenital infections Agarose gel electrophoresis [164]

24. Dermatansulfate and

heparansulfate

Intrinsic monitoring and screening tool

for mucopolysaccharidosis I patients

LC-MS/MS [165]

25. Epstein-barr virus antibodies Measurement of EBV p18-VCA antibodies

as a marker of cell-mediated

immune function

Immunofluorescence [166]

26. Estradiol, progesterone

and testosterone

Gonadal hormones measurement

for verification of menstrual cycle phase

RIA [167]

27. Folate Assay for Serum folate Microbiological assay [168]

28. Frataxin Diagnosis of Friedreich ataxia (FRDA) Immunoassay [169]

29. Follicle stimulating hormone,

luteinizing hormone, prolactin,

testosterone, estradiol,

dehydroepiandrosterone-sulfate,

androstenedione, cortisol and sex

hormone binding globulin

Longitudinal, population-based

developmental epidemiologic

study of puberty

[149]

(Continues)

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wileyonlinelibrary.com/journal/dta Copyright © 2014 John Wiley & Sons, Ltd. Drug Test. Analysis (2014)

Table 2. Continued


30. Gamma-Hydroxybutyric acid Newborn screening of succinic

semialdehyde dehydrogenase

(SSADH) deficiency

LC-MS/MS [170]

31. Gaucher disease High throughput screening method

for Gaucher disease

Fluorescent assay [171]

32. Glucocerebrosidase and

α-l-iduronidaseGaucher and Hurler disease screening

in newborn

Fluorometric enzymatic activity

assay with digital microfluidics

[172]

33. Glucose Quantification of blood glucose levels Reflectometery [173]

34. Glutathione Ultramicro method for determination

of total glutathione

Colorimetry [174]

35. Guanidinoacetate and creatine Diagnosis of guanidinoacetate methyltransferase

and arginine glycine amidinotransferase

deficiencies for newborn screening

Flow injection analysis-MS/MS [175]

36. Haemoglobin A1c A tool for the diagnosis of diabetes Immunoturbidimetricmethod [176]

37. Haemoglobin peptides Detection of numerous haemoglobinopathies LC-MS/MS [177]

38. Hepatitis A antibodies Anti-HAV antibody testing ELISA [178]

39. Hepatitis B antigen Hepatitis diagnosis PCR [179]

40. Hepatitis C virus (HCV) core

antigen and anti-HCV

antibodies (HCV AgAb)

HCV infection and diagnosis EIA [180]

41. HIV antibodies, hepatitis C antibodies,

and hepatitis B antigens

Simultaneous detection of HIV 1 and

hepatitis B and C

Multi-analyte immunoassay [181]

42. HIV-1 antibodies Simultaneous measurement of antibodies

to three HIV-1 antigens

Fluorescent immunoassay [39]

43. Luteinizing hormone and

follicle-stimulating hormone

Female gonadotropins measurement Immunofluorometric assays [182]

44. Homocysteine For diagnosis of Homocystinuria HPLC [183]

45. HTLV-I specific antibodies For detection of human T cell lymphotropic

virus infection

Modified Serodia assay [184]

46. Human immunodeficiency

virus RNA

Diagnosis of HIV infection and

monitoring of therapy

HIV-1 Monitor assay kit [185]

47. Human immunodeficiency

Virus type 1

Infant HIV infection diagnosis using

optimized Up24 antigen assay

ELAST amplification kit [186]

48. Hunter syndrome Single step diagnosis of Hunter syndrome Microfluidics [187]

49. IgE In allergic sensitization assays Immunofluorescence microscopy [188]

50. IGF-I, IGFBP-2 and IGFBP-3 To monitor changes of IGF-I, IGFBP-2 and

IGFBP-3 content in blood and assessment

of risk regarding pancreatic cancer in

the prostate, lung, colorectal and ovarian

cancer and assessing growth in childhood

RIA [189]

51. Inflammatory markers and

neurotrophins

Epidemiologic case–control studies and in

neonatal screening

Immunoassay with xMAP technology [190]

52. Insulin Early-life determinants of circulating insulin ELISA [191]

53. Interleukin-6 Diagnosis for causes and consequences

of inflammation

ELISA [192]

54. Interferon-γ inducible protein

(IP)-10

Newborn screening for M. tuberculosis infection ELISA [193]

55. Lipoprotein (LP) For quantification of LP ELISA [194]

56. LSD Enzyme For diagnosis of lysosomal enzyme disorders [195]

57. Markers for Hepatitis A and B For large scale screening programmes

of HBs Ag-carriers and most

anti-HBs-positive individuals prior

to an immunization campaign.

RIA [196]

58. Mucopolysaccharidosis type II For identification of MPS II patients Fluorescence immunoassay [197]

59. Perfluorooctanesulfonate (PFOS),

perfluorooctanoate (PFOA),

and bisphenol A (BPA)

Newborn screening on environmental

chemical exposures

LC-MS/MS [198]

(Continues)


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Drug Test. Analysis (2014) Copyright © 2014 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/dta

Table 2. Continued


60. Lymphoproliferative diseases Detection of monoclonal immunoglobulin

gene rearrangement

PCR [199]

61. Phenylalanine and tyrosine Diagnosis of phenylketonuria Capillary electrophoresis-MS [200]

62. Phenylketonuria Phenylalanine and tyrosine quantification

in neonatal blood

LC-MS/MS [201]

63. Phosphatidylethanol Monitoring of alcohol misuse LC-MS/MS [202]

64. Pompe disease For diagnosis of Pompe disease Enzyme assay [203]

65. Pregnancy- associated plasma

protein A and free β-subunit ofhuman chorionic gonadotropin

hormone

First-trimester prenatal screening

for aneuploidy

Enzyme assay [204]

66. Prolactin For diagnosis of seizures Immunoenzymatic assay [205]

67. Prostate specific antigen Prostate cancer screening Chemiluminescent Immunoassay [206]

68. Prostate specific antigen (PSA) Mass screening for prostate cancer Chemiluminescent immunoassay [206]

69. Retinol, tocopherols, beta-carotene,

vitamin C and homocysteine

Malnutrition assay in

developing countries

HPLC [207]

70. Somatomedin-c In diagnosis of pituitary gland

disorder and abnormalities in

growth hormones production

RIA [208]

71. S. pneumoniae and H. influenzae b Dignosis of Pneumonia RT-PCR [209]

72. Succinylacetone Diagnose of Tyrosinemia type 1 LC-MS/MS [210]

73. Succinylacetone, amino acids

and acylcarnitines

New born screening for

Tyrosinemia type I

FIA-MS/MS [211]

74. Tay-sachs and sandhoff disease For detection of Tay-Sachs and

Sandhoff disease

MS/MS [212]

75. Thyroglobulin Thyroid function test Fluoroimmunometric Assay [213]

76. Thyroid-antibody For detection of thyroid disease ELISA [214]

77. Thyrotropin (TSH) and Thyroxine (T4) For diagnosis of congenital

hypothyroidism

Fluorescent immunoassay [215]

78. Thyroxine For diagnosis of hypothyroidism RIA [216]

79. Thyroxine binding globulin Identification of levels changing of

thyroid hormone

RIA [217]

80. Transferrin Receptor Iron deficiency Immunoassay [218]

81. Triglycerides Diagnosis of heart diseases Enzyme assay [219]

82. Triiodothyronine (T3) Diagnosis of congenital hypothyroidism ELISA [216]

83. Thyroid stimulating hormone Diagnosis of congenital hypothyroidism ELISA [220]

84. Vitamin D3-25-OH and

Vitamin D2-25-OH

In diagnosis of hypovitaminosis D LC-MS/MS [221]

85. α1-antitrypsin genotypes Early diagnosis of hereditary disorder,

associated with early risk of onset

chronic obstructive pulmonary disease

and liver dysfunction

Fluorometric elastase

inhibition assay

[222]

86. α-galactosidase Dignosis of Fabry disease Chip-based nanoelectrospray

ionization mass spectrometry

[223]

87. α-iduronidase Mucopolysaccharidosis I diagnosis Enzyme assay [224]

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of quality control samples with different hematocrit levels canreduce the quantifiable and unacceptable deviations. [1,32]

Precautions are required in metabolomic profiling using DBS assome of the metabolites namely L-lysine, iminodiacetic acid,DL-threo-beta-hydroxyaspartic acid, citric acid, adipamide, andadenosine-5-monophosphate, are found to be absent in DBSextracts but are detected in blood or plasma. [49] Another pitfall,which is usually overlooked during analyte analysis from DBScards through LC-MS/MS, is the potential interaction betweenanalyte and card constituents which are sometimes culprits forionization suppression in the MS source and also for a change


in chromatographic retention time and peak shape distortion.Treated DBS cards consist of several types of proprietary reagentslike sodium dodecayl sulfate, tris(hydroxymethyl)aminomethane,guanidinium thiocyanate, and others, which interrupts either inionization due to their non-volatile nature resulting in suppressedionization or reduced signal intensity and often results in ion-paring with analytes leading to chromatographic peak shapedeformity and altered retention time. This necessitates carefulevaluation of possible DBS card interactions and interferences.[52,56] Moreover, DBS method validation is more error proneand time consuming in comparison to conventional plasma



Drug Testing

and Analysis

samples as estimation of several supplementary validationparameters like analyte stability on cards during drying andstorage, the effect of hematocrit, spot homogeneity, and ana-lyte elution efficiency from the card are also mandatory.

Regulatory aspects

DBS technology is well accepted worldwide for the purpose ofnewborn screening for metabolic, endocrine, hematologic, andother disorders. [57,58] The US FDA has approved the use of DBScards for in vitro testing of HIV types 1 and 2. [59] As alreadymentioned, three DBS cards have been approved by the USFDA as medical devices for blood sample collection. The WorldHealth Organization (WHO) and the Centers for Disease Controland Prevention (CDC) have established the guidelines for DBSspecimen collection, storage, and transportation. [24,26] Ampleresearch papers (Tables 1 and 2) have been published for quanti-tative analysis of several drugs using the DBS technique and itsadvantages in preclinical and clinical studies are being realized.The well-established magical 3 ‘R’ benefits of DBS technique –

replacement, refinement and reduction of animals in research– are well supported by the EBF and UK Medicines andHealthcare products Regulatory Agency. [60] The EBF has madeseveral recommendations which are intended to provide guid-ance for analysis of DBS samples and method validation. [61]

Unlike plasma, serum, or whole blood, a DBS specimen is notconsidered as an acceptable bio-matrix by regulatory bodiesfor PK studies during a drug discovery and developmentprogramme. But, further advances of this technique togetherwith exclusive benefits in conjugation with regulatory guide-lines will make it acceptable worldwide.

Conclusion

The power of modern analytical tools together with financial andethical inspirations has made DBS a high-flying blood samplingtechnique. Apart from a few limitations, the DBS samplingmethod is unquestionably the most ethical and economicaltechnique for blood sample collection, shipping, and storage.For personalized medication, it may be a boon to the medicalpractitioners by getting microbiome and metabolome profileswith single window drug monitoring and diagnosis of diseases.Modified DBS methods like perforated DBS (PDBS), bilayer DPScard, or Hemaspot technology should be encouraged to over-come the drawback of conventional DBS methods. A lot ofadvancements are still awaited to resolve the concerns ofrepeated or additional analyses, homogeneity of spot, [17,21]

effect of hematocrit value of blood, [61,62] recovery of DBS andreduction of the signal-to-noise ratio by blood cell component sep-aration. [63] Appreciation of regulatory authorities and sponsors isrequired to use the DBS technology with automation for expansionof drug discovery in shorter duration with limited resources.

Acknowledgment

Author AS is thankful to ICMR, New Delhi, for financial supportin the form of his fellowship for the CDRI Communication(8626).


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dried blood spots: concepts, present status, and future perspectives in bioanalysis

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