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Drug Testing

Review and Analysis

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

( DOI 10.1002/dta.1646

Dried blood spots: Concepts, present status,

and future perspectives in bioanalysis
Abhisheak 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 qualitative
and quantitative bioanalysis context. In the DBS method, the blood sample is directly soaked on to a paper (with or without
treatment). After drying it can be analyzed by modern analytical, immunological, or genomic detection systems. Several
advantages 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 analysts
to biohazards, make it the most appropriate blood sampling technique. This review illustrates the information available on
the DBS method which may serve as a single window for investigators in the eld of bioanalysis. Also, it explores the
prociency 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

Introduction technique, public health laboratories screened more than 95% of

all newborns in the USA for inborn metabolic disorders. [5] For PK
Implementation and effectuation of dried blood spot (DBS) and TK studies in small animals, such as rats or mice, the DBS
technology in the realm of therapeutics and health sciences has method can facilitate rich sampling and these samples are stable
simplied the conventional blood collection and analysis process. at a wide range of temperatures. This technique has been reported
DBS is anticipated to be a promising surrogate of established liquid to be suitable for drugs which are susceptible to photodegradation.
bio-matrices (plasma/serum) for pharmacokinetic (PK) and Forced and natural photodegradation experimental data has shown
toxicokinetic (TK) studies and could even surpass them. Ivar Chris- that nifedipine and omeprazole exhibit higher photostability when
tian Bang, the father of Modern Clinical Microchemistry, pioneered spotted and stored on various DBS paper than that in water, plasma,
the DBS method for quantication of blood sugar.[1] After half a cen- or whole blood. [6] DBS-assisted analysis has extensive applications
tury of Bangs DBS application, [1] Guthrie and Susi [2] in 1963, in PK and TK studies, therapeutic drug monitoring (TDM), disease
reported the DBS method for the analysis of phenylketonuria in ne- screenings, testing of doping substances and metabolomic studies.
onates and thereafter the DBS method gained popularity in It is being widely utilized in the pharmaceutical industries, hospitals
bioanalysis. Blood is the most preferred and regulatory acknowl- and research centers, particularly where blood or plasma sample
edged biological sampling matrix for in vivo concentration assess- volumes are low, difcult to collect, store, process or transport.
ment in variety of species. [3,4] Venipuncture, the traditional blood
sampling technique, has a major drawback of being an invasive
and painful technique. In clinical trials, venipuncture may be Tools and techniques
responsible for poor volunteer recruitment. Moreover, serial
sampling is difcult in small animals during PK and TK studies due Analysis of component(s) (exogenous and/or endogenous) in bio-
to the requirement of large blood volume withdrawal. Unlike the logical matrix by the DBS sampling method involve following steps:
downsides of venipuncture, blood sampling through DBS is
minimally invasive (nger prick versus venipuncture in humans; Selection of paper
blood collection through tail vein versus retro-ocular or intra-cardiac
DBS cards are composed of non-cellulose or cellulose (lter paper)
in laboratory animals). Following the collection of blood drops on a
matrix of specic pore size and thickness. Nowadays, various
paper, drying, and transportation, the blood spot is extracted and
commercial DBS cards are available, namely Whatman 903 cards,
analyzed in the laboratory. DBS cards are considered to be less
biohazardous in comparison to plasma samples in view of a fact
that blood is in dried form; all the proteins, pathogens, and enzymes * Correspondence to: Jawahar Lal, Pharmacokinetics & Metabolism Division,
are inactivated on the card and bacterial growth is prevented. The CSIR-Central Drug Research Institute, Jankipuram Extension, Sitapur Road,
less invasive nature of DBS and the use of micro-volume blood Lucknow - 226031, India. E-mail:
samples have made it a very useful sampling method for neonatal
a Pharmacokinetics & Metabolism Division, CSIR-Central Drug Research Institute,
and juvenile subjects. DBS reduces the number of steps preceding Lucknow - 226031, India
drug analysis (i.e. requirement of cold chain during transportation
and storage as well as centrifugation) (Figure 1). By using the DBS b Academy of Scientic and Innovative Research, New Delhi, India

Drug Test. Analysis (2014) Copyright 2014 John Wiley & Sons, Ltd.
Drug Testing
and Analysis A. Sharma et al.

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

FTA DMPK type-A, B, C cards and FTA Elute cards (GE Healthcare, unknown blood volume spotting on conventional DBS cards, Gabriel
Piscataway, NJ, USA), as per the type of analytical requirements. et al. [12] developed a disposable chip. It has self-actuated dissolvable
Routinely, Whatman 903 cards are basically used in newborns valves which meter and transfer exact volume of the blood in
screening, FTA DMPK type A, B, C cards are used in PK/TK studies microlitres.
and FTA Elute cards are intended mainly for collection and puri-
cation of DNA for downstream analysis. All types of DMPK cards
Specimen collection
are available in two forms: regular and indicating. Indicating cards
are useful for colourless samples like urine, plasma, synovial uid, For TDM and diagnosis of diseases in humans, whole blood
and cerebrospinal uid. DMPK type A and B cards are chemically sample is collected from a nger, toe, or heel prick with the aid
treated with proprietary reagents that, on contact cause lyses of of sterile disposable lancet or surgical blade. For PK and TK
cells, denature proteins, inactivate enzymes, and prevent the studies in rat and mouse, blood can be collected from the caudal
growth of bacteria. These coated cards are prepared to cause lyses vein. For qualitative purpose, the DBS specimen can be prepared
of both cellular and nuclear membranes to expose nucleic acids by cautiously applying a few freshly drawn blood drops from a
with good stability for storage and analysis. [7] These DMPK cards nger/toe prick on a card. When the objective is quantitative
also inhibit the enzymatic degradation of several analytes namely analysis, a measured amount of blood volume is gently applied
procaine and acetyl salicylic acid from esterases which are present on a DBS card with the aid of a pipette or capillary tube. In such
in the blood. These enzymes are denatured and inactivated when cases, use of an anticoagulant is a perquisite for appropriate
blood is spotted on the card leading to enhanced analyte stability. spotting. Although heparin or ethylenediaminetetraacetic acid
DMPK-C and Ahlstrom 226 cards (ID Biological Systems, Green- (EDTA) can be used as an anticoagulant, the latter is more
ville, SC) are not treated with any chemical; therefore, there are no suitable. However, EDTA may cause interference in MS but it has
impregnated chemicals to interfere with the analysis. Moreover, advantages over heparin in its mixing and drying abilities along
proteins will not be denatured thus DMPK-C and Ahlstrom 226 with calcium-dependent phospholipases and ester hydrolases inhi-
cards may be better choice for protein based biomolecules analysis. bition. [1315] Use of anticoagulants signicantly affects the results in
US Food and Drug Administration (FDA) has approved three DBS telomere length measurements in quantitative polymerase chain
cards, namely Ahlstrom 226-K062932, Whatman 903 and PerkinElmer reaction (qPCR) based analysis in DBS. [16]
226 under 21 CFR 862.1675 as medical device for blood specimen col- Volume of blood to be spiked on the DBS card depends on the
lection. [9] Non-cellulose DBS cards (Bond Elut DMS Card, Agilent sensitivity of bioanalytical method or instrumentation facility. The
Technologies, Santa Clara, CA, USA) are also commercially available pipette tip should be held just above the card allowing drop
for DMPK research. They are claimed to be superior in form of im- formation and soaking onto the surface. The pipette tip should
proved mass spectrometry (MS) signal, less effort in punching and he- not be touched repeatedly to the surface of the card as it
matocrit independent spot homogeneity. [10] In-house treatment of may damage the paper leading to sample inhomogeneity.
cards has also been reported for compound specic stability. Guowen Multi-layered DBS spot formation may occur due to repeated
et al. [11] used citric acid solution on a DBS card to stabilize their drug application of blood drops in the same collection circle. All these
candidate (KAI-9803) from thiol-disulde exchange. Irrespective of should be avoided as they make the specimen invalid and lead to Copyright 2014 John Wiley & Sons, Ltd. Drug Test. Analysis (2014)
Drug Testing
An overview on dried blood spots and Analysis

misinterpretation of results. Improperly labelled cards as well as ambient temperature. For protection from environmental humid-
dark, supersaturated, clotted, discoloured, contaminated, and ity, DBS cards are advised to be carefully wrapped and packed in
serum rings containing spots are considered as invalid specimens. sealable heavy duty plastic bags with adequate desiccant and a
As described by Ren et al., [17] autoradiography may be a solution humidity indicator to nd out at what time the desiccant has to
for visualization of uneven distribution of analyte(s) on DBS spots. be replaced. DBS cards are considered as non-regulated and
Hematocrit value, which represents the measure of packed cell vol- exempt material as per US Department of Transportation (DOT)
ume in blood, affects the size of DBS and is usually the main cause and the US postal service. Properly labelled DBS cards packets,
of the bias in results. Blood samples with different hematocrit value which clearly convey the biohazardous nature of the content
possess variable viscosity and spreadability on the card resulting in inside package to transportation personnel and other employees,
dissimilar spot size. In such situations, conventional assumption of can be shipped to analytical laboratories through mail, courier, or
direct proportionality relationship between sample volume and express mail delivery services. For establishing sample integrity
spot size fails. Hence, xed spot area measurement cannot be and safety from occupational exposure of hazardous blood
applied for quantitative purposes. Also, there are a few other factors samples, basic triple packaging technology is used for DBS card
such as the type of card material and the environment of spotting shipment. Triple package comprises of primary container, sec-
which contribute to the uneven distribution of analyte on DBS ondary container, and a third covering of high quality paper
cards. Several approaches were tried to overcome the effects of envelope with an afxed or printed version of the international
these factors. [18] Blood samples can be spotted for a pre-cut dried biohazard symbol. DBS packages can be stored at cool and dry
blood spots (PCDBS); this will nullify the effect of the hematocrit by place as such or can also be kept in polystyrene foam boxes until
extracting the whole spot without punching a specic area. [19] Use transportation to laboratories. If long-term stability of certain
of capillaries with xed volume dispenser for nger pricking and analytes at room temperature is not established on DBS cards,
development of novel cards with less propensity to impact of he- the packed DBS cards with desiccant can be stored in laboratory
matocrit and drying milieu, may be implemented. [18] Capiau et al. freezers until analysis to minimize analyte degradation. [2427]
reported a method for determining the hematocrit value in
any DBS by measuring potassium in the DBS extract using a
Addition of internal standard (IS)
routine clinical chemistry analyzer. This add-on technique can
assess the haematocrit-dependent intra-individual variability and Addition of IS in the DBS method is a tricky task. In most of the cases,
further facilitate non-volumetric DBS sampling. IS is added before extraction, either directly in extraction tube or in
extraction solvent. In such circumstances, IS does not provide the ex-
traction efciency from the DBS spot and act as an external stan-
Drying of the card
dard. [28] In a second method, IS can be pre-spotted on DBS card
After sample collection, the DBS cards are generally dried before addition of blood sample for better results. [29] IS can also
horizontally for 23 h on a card rack at room temperature or be sprayed on a DBS card for homogeneity across the spot
under nitrogen ow and controlled humidity. [21] The time ensuring reproducibility. [28] Spraying or spotting of IS on a
required for drying the card will depend on the type of card as DBS card is a worthwhile method compared to the addition of
well as the sample volume. Care should be taken not to lay a IS in extraction tubes or extraction solvent, as the latter is incapa-
hand on wet spots. Cards should not be stacked or allowed to ble of nding extraction variability of analyte(s) from the DBS.
touch other surfaces while drying. Exposure of the DBS specimen
to any milieu like direct sunlight, dust, or ying insects should be
Extraction and analysis
avoided as that may compromise its integrity. In most of the
research papers, analytes were reported to be stable after drying Dried cards can be punched out with various available diameter
and in between analysis tenure. However, drying of the card is punching tools (manual, semi-automated, and automated).
the crucial step for unstable analytes. Various modications like Punched dried cards can be used directly (by microuidics) or
changes in pH, [11] temperature, and humidity are recommended. by extraction of analytes with suitable extraction solvent. Extrac-
Heat stabilization can also be a solution for metabolically tion solvent should be optimized as per the solubility prole of
unstable drugs. According to Blessborn et al., [23] enzymatic the analyte(s) with consideration of minimizing extraction of
activity of blood can be reduced by drying DBS card at 95 C for interfering endogenous impurities. Extraction efciency from xed
30 s. They found that oseltamivir, cefotaxime, and ribavirin are sta- DBS can be improved by addition of liquid ammonium. [30] After ex-
ble after heat stabilization but artemether and dihydroartemisinin traction, samples are subjected to analysis. Liquid chromatography-
were unstable due to their decomposition at 60 C. StabilizerTM tandem mass spectrometry (LC-MS/MS), desorption electrospray
instrument from Denetor (Gothenburg, Sweden) is commercially ionization mass spectrometry (DESI-MS), gas chromatography
available for heat stabilization of DBS cards. After drying, these mass spectrometry (GC-MS), matrix assisted laser desorption mass
cards are suitable for analysis, transportation, or storage. spectrometry (MALDI-MS), MALDI time-of-ight mass spectrometry
(MALDI-TOF-MS), high performance liquid chromatography (HPLC),
isoelectric focusing (IEF)-HPLC, direct laser desorption (LD) TOF-MS,
Storage and transportation
inductively coupled plasma mass spectrometry (ICP-MS), laser
In contrast to conventional biological matrices, DBS provides a ablation (LA) ICP TOF-MS, polymerase chain reaction (PCR), enzyme
huge simplication in the arena of storage and transportation. linked immunosorbent assay (ELISA) and microuidic chip have suc-
Unlike plasma/serum samples, DBS requires neither freezers for cessfully been coupled with the DBS method for qualitative and quan-
storage nor bulky volumes of dry ice for shipping which are costly titative analyses of blood samples. [13] An extraction-free direct spray
and need special care. Barring the humidity factor, which has sig- ionization technique is reported by Manicke et al. [31] in which the an-
nicant inuence on specimen stability and elevates the chances alyte is converted into gas phase ions using solvent electrospray
of bacterial growth, DBS cards can be shipped and stored at by applying a high voltage (3500 V) to the wet substrate.

Drug Test. Analysis (2014) Copyright 2014 John Wiley & Sons, Ltd.
Drug Testing
and Analysis A. Sharma et al.

Automation DBS applications

Commercial instruments are available for fully automated online Application of DBS technology has gained momentum in various
DBS sampling and analysis. Online automated tools (ABS2; Instech elds including neonatal metabolic screening, TDM, preclinical and
Solomon, Plymouth Meeting, PA, USA and Culex; BASi, West clinical PK, TK, forensic, biological, and immunological sciences.
Lafayette, IN, USA) are capable of collecting blood from freely Literature reports on the applications of DBS in the investigations
moving laboratory animals and can be coupled for serial sampling of drug, biomarkers or diseases are compiled in Tables 1 and 2.
(in microlitre of blood volume) on DBS cards with high throughput
and accuracy. Automated Sample Card and Prep (SCAP) system
(Prolab, Reinach, Switzerland) can be coupled with LC-MS/MS for Preclinical pharmacokinetic/toxicokinetic study
online drug analysis. Ganz et al. [32] applied this system for the on- Serial blood micro-sampling is possible with DBS in rodents
line analysis of bosentan and its three metabolites in human DBS without changing their haemodynamic balance. By implementing
with adequate accuracy and precision. Deglon et al. [33] have the DBS method, animal requirements can be reduced to the
innovated an online analytical method in which sample can be extent of at least 60%. Preclinical PK studies in rodents require
analyzed by online desorption of DBS spot in an inox cell by sampling of blood with 12 mL per sample at multiple time-
column switching mode with assurance of purication and points. Therefore, the studies are done by sparse sampling. Thus,
separation of samples. This online method paves a way to high the low sampling volumes required for DBS also facilitates serial
throughput by reducing various steps of bioanalysis like sampling from the same animal, offering improved data quality
centrifugation, extraction, vortexing, drying, or reconstitution. over composite sampling paradigms from multiple study animals.
Additionally, it will reduce the cost of experiment by reducing the
Calculation and correlation with plasma/serum results number of animals especially in the case of transgenic or
knockout model studies. Also, it eliminates the harvesting step
Blood level of drug is the sum of levels of drug in red blood cells of serum/plasma and reduces the quantity of new chemical
(RBCs) and plasma. Often due to differential binding of drug to entities (NCE) required during drug discovery and development
specic blood components, concentration in plasma and RBCs programme. Dainty et al. [36] have compared the DBS method with
varies; hence considering whole blood concentration as equiva- whole blood results and reported that the results from both are
lent to plasma concentration becomes inappropriate. Before similar in relation to drug concentration-time prole, area under
comparing the plasma/serum levels with DBS levels, parameters curve (AUC), stability, precision, variability and data accuracy. For
like hematocrit (H) and RBC to plasma partitioning (KRBC/plasma) the compounds with high RBC to plasma partition coefcient, using
or blood-plasma partitioning (KBlood/plasma) should be considered plasma as an analytical matrix leads to signicant errors in pharma-
as DBS results may differ from serum or plasma results. Hemato- cokinetic-pharmacodynamic (PK-PD) calculation due to haemolysis
crit is calculated by dividing packed RBC volume by total blood of blood. These errors can be overcome by replacing plasma with
DBS. [37] Liang et al. [38] have successfully applied the DBS method
volume and packed RBC volume can be determined simply by
for discovery PK study of multiple NCE by cassette dosing.
centrifugation of heparinized blood in microhematocrit tubes at
10 000 rpm for 5 mim. A conventional method for estimating
KRBC/plasma or KBlood/plasma involves spiking of drug in plasma/ Therapeutic drug monitoring
plasma water containing suspended RBCs, followed by equilibra- TDM is the quantication of drug(s) concentration in patients
tion, centrifugation and then quantication of drug concentra- blood with time to optimize the dose regimen. It helps the
tions in plasma/plasma water and RBCs. [34] Once these clinician as well as the patient regarding drug safety, as dose
parameters are known, plasma concentration can be calculated optimization can be achieved without compromising the efcacy
from DBS results by using the following equation. of therapeutically potent molecules. TDM may help to minimize
the incidences of drug resistance as antibiotic or antiviral drug
concentration levels can be maintained within the therapeutic
window. As shown in Table 1, several methods are reported for
Analyte concentration in DBS TDM using DBS as a sampling technique. The DBS method is
Calculated plasma concentration h i
1-H H*KRBC=plasma especially suitable for TDM in neonates. It can also be used for
establishing TDM units in developing countries to save the initial
(1) investment as well as processing cost of test samples.

If KRBC/plasma or KBlood/plasma is:

Population disease control and clinical pharmacokinetic
1. greater than one, DBS will have higher drug levels than plasma study
or serum results and DBS will have better PK/PD correlation.
2. equal to one, DBS results should be identical to plasma or Multi-centric clinical investigations can be studied easily with the
serum results and DBS can be an alternate for plasma study. help of the DBS technique. Congenital or genetic disorders in ne-
3. less than one, DBS results should have lower analyte levels than onates are being investigated by the DBS method. Many diseases
plasma or serum. In this condition, the DPS (dry plasma spot) can be screened by using the DBS sampling technique (Table 2).
method can be applied with two layered polymer lm which The toe or nger prick is always better than venipuncture as it
separates plasma from whole blood without centrifugation. [35] can improve volunteer recruitment in clinical studies. DBS is al-
ways advantageous for multiple sampling in remote areas where
DBS is the best suitable method for sampling of drugs having transportation, cold chain facilities, or intravenous blood sam-
higher afnity to RBC in comparison to plasma. pling may reduce the efciency of mass policy implementation. Copyright 2014 John Wiley & Sons, Ltd. Drug Test. Analysis (2014)
Drug Testing
An overview on dried blood spots and Analysis

Table 1. Applications of DBS for investigation of drugs

S. No. Drug name Application Analytical technique Reference
1. Acetaminophen TK studies LC-MS/MS
2. Actinomycin-D PK studies LC-MS/MS
3. Amodiaquine, chloroquine Preclinical PK studies LC-MS/MS
and chlorthalidone
4. Amprenavir TDM and PK studies LC-MS
5. Antiepileptic drugs (AEDs) [levetiracetam, TDM HPLC
lamotrigine, phenobarbital,
carbamazepine and
its active metabolite
carbamazepine-10,11 epoxide]
6. Atazanavir TDM and PK studies LC-MS
7. Atenolol TDM and PK studies LC-TOF-HRMS
8. Bisoprolol, ramipril and simvastatin TDM LC-HRMS
9. Bosentan PK studies LC-MS/MS
10. Busulfan TDM LC-MS/MS
11. Caffeine PK studies LC-MS/MS
12. Canrenone Neonatal screening LC-MS
13. Centchroman and 7-demethylated PK and drug-drug interaction study LC-MS/MS
14. Chloroquine and desethylchloroquine PK studies HPLC-UV
15. Choline theophyllinate PK studies Fluoroimmunoassay
16. Clonidine Preclinical and clinical PK studies LC -MS/MS
17. Clozapine TDM HPLC
18. Cocaine Control of subjects suspected of HPLC coupled with
driving under the inuence spectrouorimetric detection
of psychotropic substances.
19. Cyclosporin A TDM Radioimmunoassay
20. Cyclosporin A and tacrolimus TDM LC-MS/MS
21. Darunavir, etravirine, raltegravir TDM LC-MS/MS
and ritonavir
22. Darunavir, saquinavir, atazanavir, TDM and PK studies LC-MS/MS
amprenavir, lopinavir, ritonavir,
etravirine, efavirenz and nevirapine
23. Dasatinib Metabolite proling LC-MS/MS
24. Dexamethasone Neonatal PK studies LC-MS
25. Dextromethorphan and dextrorphan Enantioselective Method LC-MS/MS
26. Diazepam PK studies LC-MS/MS
27. Donepezil Preclinical PK studies LC-MS/MS
28. Efavirenz TDM RP-HPLC-UV
29. Ertapenem TDM and PK studies LC-MS/MS
30. Estradiol Clinical studies RIA
31. Ethyl glucuronide (EtG) and Diagnosis of recent alcohol uptake LC-MS/MS
ethyl sulfate (EtS)
32. Etravirine TDM and pharmacological research LC-MS/MS
33. Everolimus TDM LC-MS/MS
34. Exenatide Clinical PK studies ELISA
35. Exendin-4 PK and TK studies LC-MS/MS
36. Fluoxetine, noruoxetine, TDM, toxicological analysis NICI-MS-MS
reboxetine and paroxetine and PK studies
37. Flurbiprofen PK studies LC-MS/MS
38. Gabapentine Preclinical and clinical PK studies LC-MS/MS
39. Gemioxacin Preclinical PK studies HILIC with
uorescence detector
40. Guanfacine Clinical PK studies LC-MS/MS
41. Linezolid TDM LC-MS/MS
42. Lopinavir TDM and PK studies LC-MS


Drug Test. Analysis (2014) Copyright 2014 John Wiley & Sons, Ltd.
Drug Testing
and Analysis A. Sharma et al.

Table 1. Continued
S. No. Drug name Application Analytical technique Reference
43. Losartan Preclinical PK studies LC-MS/MS
44. Metformin TDM HPLC-UV
45. Metformin and sitagliptin PK studies LDTD-MS/MS
46. Methadone TDM HPLC
47. Metoprolol Preclinical, clinical PK LC-MS/MS
and doping studies
48. Metronidazole PK/PD studies in neonatal patients HPLC
49. Midazolam PK studies LC-MS/MS
50. Monodesethylchloroquine, chloroquine, PK and epidemiological studies HPLC
cycloguanil and proguanil
51. Morphine and 6- acetylmorphine Forensic study LC-MS/MS
52. Moxioxacin TDM LC-MS/MS
53. Mycophenolic acid TDM and post-marketing LC-MS/MS
clinical studies
54. Naproxen Bioavailability and PK studies LC-MS/MS
55. Nevirapine and Efavirenz TDM LC-MS/MS
56. Nifedipine Photodegradation experiments LC-MS/MS
57. Non-conjugated testosterone, testosterone Sports drug testing / dope testing GC-MS
glucuronide (TG), androsterone glucuronide
(AG) and etiocholanolone glucuronide (EtG)
58. Nofovir and emtricitabine PK studies LC-MS/MS
59. Omeprazole PK studies LC-MS/MS
60. Opiates, cocainics and amphetamines Illicit drugs determination LC-MS/MS
in suspected cases of
driving under the inuence
of drugs
61. Oseltamivir Pediatrics TDM and PK studies LC-MS/MS
62. Paclitaxel Preclinical PK studies LC-MS/MS
63. Peginesatide Sports drug testing LC-MS/MS
64. Pioglitazone TK studies LC-MS/MS
65. Posaconazole Clinical PK studies LC-MS/MS
66. Pramipexole Preclinical PK studies LC-MS/MS
67. Propranolol PK studies in neonates or LC-MS/MS
young infants
68. Quinine and 3-hydroxyquinine Clinical PK studies HPLC with
uorescence detection
69. Raltegravir TDM and pharmacological research LC-MS/MS
70. Ramoplanin Clinical PK and bioequivalence studies LC-MS/MS
71. Ranitidine TDM in pediatrics samples LC-MS/MS
72. Rifampicin TDM HPLC-UV
73. Rifaximin TDM, assessing adherence to LC-MS
medications and preventing
toxicity in a clinical setting
74. Rosiglitazone PK studies LC-MS/MS
75. Runamide TDM LC-MS/MS
76. Salmeterol TDM HPLC
77. Sisomicin and netilmicin TDM in geriatric and paediatric patients HPLC with pre-column
derivatization and
uorimetric detection
78. Sulfadoxine and pyrimethamine TDM and epidemiological studies HPLC-UV
79. Sulfadoxine and sulfamethoxazole HPLC-UV
80. Tacrolimus TDM LC-MS/MS
81. Tacrolimus and creatinine TDM and assessment of renal function LC-MS/MS
82. Tacrolimus, sirolimus, everolimus and TDM LC-MS/MS
cyclosporin A
83. Tafenoquine TDM HPLC with uorescence [136]


(Continues) Copyright 2014 John Wiley & Sons, Ltd. Drug Test. Analysis (2014)
Drug Testing
An overview on dried blood spots and Analysis

Table 1. Continued
S. No. Drug name Application Analytical technique Reference
84. Theophylline TDM, particularly suited for domiciliary care Fluorescence polarization
immunoassay (FPIA)
85. Topiramate TDM FPIA and LC-MS/MS
86. Vincristine Clinical pharmacological studies LC-MS/MS
87. Zatebradine PK studies LC-MS/MS
88. Zidovudine Monitoring of zidovudine therapy among RIA
HIV-infected pregnant mothers and their new borns
89. Zopiclone Forensic studies LC-MS/MS

DNA-based diagnosis of infectious and genetic disorders can be Limitations of DBS technique
performed through DBS-coupled PCR tests such as human
cytomegalus virus (HCMV) detection, diagnosis of HIV, T-cell re- Although the DBS technique has gained massive success in the
ceptor excision circle (TREC), Fabry disease mutation and spinal arena of bioanalysis and experienced a surge of interest in several
muscular atrophy (SMA) diagnosis, etc. [13,3943] elds, a plethora of questions exist whose answers are up in the
In the USA, the DBS method was evaluated and validated by air. The most distinct advantage of the DBS is the use of low
centres for disease control and prevention for new born screen- blood volume which is correspondingly a limitation of the DBS
ing tests. The DBS method has also been reported for a plethora method as it can only be coupled with highly sensitive analytical
of analytes, biological markers, nutrients, antibodies, and en- techniques. It is not as amenable as plasma or serum samples
zymes for diagnosis and monitoring of epidemiological diseases. with the most common analytical instrument like HPLC coupled
Snijdewind et al. [45] especially recommended use of the DBS with UV-Visible detector. Also, the DBS sampling technique is in-
method in epidemic surveillance, screening, and follow-up of vi- adequate for air sensitive or volatile analytes. [21,52] Basically, DBS
ral infection and its treatment in remote areas. technology involves use of capillary blood from volunteers in
Conducting a clinical trial is always a costly affair. In case of a clinical studies; however, there are plausible chances that capil-
worldwide multi-centric clinical study, cold chain (freezer or dry lary blood analyte concentration may vary from venous blood.
ice) supported transportation of biological samples from the clin- In that case, DBS results may differ from those obtained with tra-
ical facility to the analytical laboratory involves huge capital in- ditional venous sampling leading to less appropriateness of this
vestment along with stability issues. After drying, spotted DBS technique for such analytes. It has been reported that paraceta-
can be transported at ambient temperature. The prociency of mol has signicantly higher concentrations in nger-prick blood
DBS can reduce the overall clinical trial budget by up to 50%. [46]
than venous blood. [53,54] For the calculation of bioavailability, se-
lection of the bio-matrix is an essential task. In case of low clear-
Other applications ance and saturable blood cell binding of the compounds, the
blood cell to unbound plasma concentration ratio will vary with
DBS is applicable to most of the blood-related qualitative or concentration. As a result, whole blood or DBS will not be able
quantitative analysis. Doping analysis of banned substances can to calculate accurate bioavailability as the blood ratios of AUCs
easily be performed with the help of DBS technology. Sports will differ. Also, DBS is not a worthy technique for compounds
event grounds or training facilities are not always near an analyt- having minimal blood cell uptake (KBlood/plasma 0.55). [37] DPS
ical laboratory. By using the DBS sampling technique, blood sam-
can be a solution in such situation. Multi-layer spotting and selec-
ples from sports personnel can easily be collected, transported,
tion of punching site on DBS card is the general cause of inaccu-
and analyzed to get unbiased results in a cheap, fast, automated,
rate results in quantitative analysis. This problem can be solved
discrete, and minimal invasive mode. Thomas et al. [47] analyzed
by using whole blood spot with xed volume for analysis rather
26 prohibited compounds in a single analysis using DBS coupled
than a specic punched part of DBS. There are chances that the
with LC-MS/MS consisting of a quadrupole mass lter, higher col-
analyte level in certain test samples could cross the calibration
lision dissociation (HCD) cell, and an orbitrap detector. DBS is not
just a technique for whole blood analysis; it can also be used for limit. Then, plasma or serum test samples can be diluted with di-
analysis of banned substances in urine or saliva [48] and lution integrity testing which is a tricky and tedious task with
implemented for dope testing. The DBS method is equally appli- DBS. Hematocrit value inuences the spread of blood on the
cable for plasma or serum analysis in medico-legal and forensic DBS card and partially inuences the results with signicant in-
applications. Kong et al. [49] performed metabolomic proling of ter-subject variability. Apart from hematocrit, spot homogeneity
695 detectable and 137 identiable markers through DBS using and compound degradation are other problems. The European
GC-MS and recommended it as a substitute of plasma for Bioanalysis Forum (EBF) has expressed concern on changes in
metabolomic studies. Lal et al. [50] used the DBS method for PK extraction recovery of analyte(s) after the aging of the DBS card.
drug-drug interaction study of centchroman and its metabolite In long-term stability studies of DBS cards, concentration varia-
with carbamazepine, indicating its applicability in PK drug-drug tion in relation to storage period has also been reported for
interaction study. Wijnen et al. [51] applied the DBS method for amino acids. It is reported that carnitine levels increased by
DNA isolation in pharmacogenetics and found that the DNA iso- 7.6% per year for rst ve years, followed by a 1.4% decrement
lation from the DBS technique is faster, cheaper, and easier than per year. [55] Using a non-cellulose DBS card, long-term stability
commercially available DNA isolation kits. studies assessment, analytical method modication, or addition

Drug Test. Analysis (2014) Copyright 2014 John Wiley & Sons, Ltd.
Drug Testing
and Analysis A. Sharma et al.

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

S. No. Drug/ biomarker/ disease Application Analytical technique Reference
1. 17- hydroxypregnenolone and Newborn screening for congenital LCMS/MS
17-hydroxyprogesterone adrenal hyperplasia (CAH)
2. 17-hydroxyprogesterone Diagnosis of neonatal CAH Microwave-assisted silylation
3. 25-hydroxyvitamin D3 Neonatal screening LC-MS/MS
4. Acid beta-D-glucosidase, acid Diagnosis of Gaucher and Enzyme analysis
sphingomyelinase, Niemann-Pick patients
chitotriosidase and alpha-N-
5. Adenosine-deaminase defect Adenosine and 2-deoxyadenosine testing MS/MS
6. Adrenal steroids Diagnosis of neonatal CAH LC-MS/MS
7. Alloisoleucine Newborn screening for Maple Syrup LC-MS/MS
Urine Disease (MSUD)
8. Androstenedione Home monitoring of steroid replacement Fluorometric immunofunctional
therapy in congenital adrenal hyperplasia. assay
9. Apolipoprotein A-I Time and temperature of storage of samples ELISA
and standards necessary for Apo A-I assays
using ELISA methodology
10. Apolipoprotein b Screening programme for new ELISA
borns as lipid metabolism
in neonates.
11. Babesiagibsoni (asian For mass screening of canine PCR
genotype) infection babesiosis in dogs
12. Biotinidase Lysosomal storage diseases (Pompe, Fabry, Microuidics
Gaucher, Hurler and Hunter)
13. C26:0-lysophosphatidylcholine X-linked adrenoleukodystrophy LC-MS/MS
newborn screening
14. Carnitine and acylcarnitines Clinical diagnostic and public health LC-MS/MS
newborn screening
4+ [156]
15. Cd lymphocytes For Cd4+ lymphocytes counting in ELISA
HIV patients
4+ [157]
16. Cd t-cell CD4+ lymphocyte counting for ELISA
the monitoring of antiretroviral treatment
17. Ceruloplasmin Newborn screening for Wilsons disease LC-MS/MS
18. Citrulline Severity screening for enteral disorders LC-MS/MS
19. Cortisol, 4-androstene-3,17-dione Screening for congenital adrenal hyperplasia LC-MS/MS
and 17-hydroxyprogesterone
20. Cotinine Biomarker of maternal smoking LC-MS/MS
21. Cotinine and trans Screening to assess second-hand smoke LC-MS/MS
3-hydroxycotinine (SHS) exposure in children
22. C-reactive protein Diagnosis of cardiovascular disease Enzyme immunoassay (EIA)
23. Cytomegalovirus infection Early diagnosis of viral congenital infections Agarose gel electrophoresis
24. Dermatansulfate and Intrinsic monitoring and screening tool LC-MS/MS
heparansulfate for mucopolysaccharidosis I patients
25. Epstein-barr virus antibodies Measurement of EBV p18-VCA antibodies Immunouorescence
as a marker of cell-mediated
immune function
26. Estradiol, progesterone Gonadal hormones measurement RIA
and testosterone for verication of menstrual cycle phase
27. Folate Assay for Serum folate Microbiological assay
28. Frataxin Diagnosis of Friedreich ataxia (FRDA) Immunoassay
29. Follicle stimulating hormone, Longitudinal, population-based
luteinizing hormone, prolactin, developmental epidemiologic
testosterone, estradiol, study of puberty
androstenedione, cortisol and sex
hormone binding globulin

(Continues) Copyright 2014 John Wiley & Sons, Ltd. Drug Test. Analysis (2014)
Drug Testing
An overview on dried blood spots and Analysis

Table 2. Continued
S. No. Drug/ biomarker/ disease Application Analytical technique Reference
30. Gamma-Hydroxybutyric acid Newborn screening of succinic LC-MS/MS
semialdehyde dehydrogenase
(SSADH) deciency
31. Gaucher disease High throughput screening method Fluorescent assay
for Gaucher disease
32. Glucocerebrosidase and Gaucher and Hurler disease screening Fluorometric enzymatic activity
-l-iduronidase in newborn assay with digital microuidics
33. Glucose Quantication of blood glucose levels Reectometery
34. Glutathione Ultramicro method for determination Colorimetry
of total glutathione
35. Guanidinoacetate and creatine Diagnosis of guanidinoacetate methyltransferase Flow injection analysis-MS/MS
and arginine glycine amidinotransferase
deciencies for newborn screening
36. Haemoglobin A1c A tool for the diagnosis of diabetes Immunoturbidimetricmethod
37. Haemoglobin peptides Detection of numerous haemoglobinopathies LC-MS/MS
38. Hepatitis A antibodies Anti-HAV antibody testing ELISA
39. Hepatitis B antigen Hepatitis diagnosis PCR
40. Hepatitis C virus (HCV) core HCV infection and diagnosis EIA
antigen and anti-HCV
antibodies (HCV AgAb)
41. HIV antibodies, hepatitis C antibodies, Simultaneous detection of HIV 1 and Multi-analyte immunoassay
and hepatitis B antigens hepatitis B and C
42. HIV-1 antibodies Simultaneous measurement of antibodies Fluorescent immunoassay
to three HIV-1 antigens
43. Luteinizing hormone and Female gonadotropins measurement Immunouorometric assays
follicle-stimulating hormone
44. Homocysteine For diagnosis of Homocystinuria HPLC
45. HTLV-I specic antibodies For detection of human T cell lymphotropic Modied Serodia assay
virus infection
46. Human immunodeciency Diagnosis of HIV infection and HIV-1 Monitor assay kit
virus RNA monitoring of therapy
47. Human immunodeciency Infant HIV infection diagnosis using ELAST amplication kit
Virus type 1 optimized Up24 antigen assay
48. Hunter syndrome Single step diagnosis of Hunter syndrome Microuidics
49. IgE In allergic sensitization assays Immunouorescence microscopy
50. IGF-I, IGFBP-2 and IGFBP-3 To monitor changes of IGF-I, IGFBP-2 and RIA
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
51. Inammatory markers and Epidemiologic casecontrol studies and in Immunoassay with xMAP technology
neurotrophins neonatal screening
52. Insulin Early-life determinants of circulating insulin ELISA
53. Interleukin-6 Diagnosis for causes and consequences ELISA
of inammation
54. Interferon- inducible protein Newborn screening for M. tuberculosis infection ELISA
55. Lipoprotein (LP) For quantication of LP ELISA
56. LSD Enzyme For diagnosis of lysosomal enzyme disorders
57. Markers for Hepatitis A and B For large scale screening programmes RIA
of HBs Ag-carriers and most
anti-HBs-positive individuals prior
to an immunization campaign.
58. Mucopolysaccharidosis type II For identication of MPS II patients Fluorescence immunoassay
59. Peruorooctanesulfonate (PFOS), Newborn screening on environmental LC-MS/MS
peruorooctanoate (PFOA), chemical exposures
and bisphenol A (BPA)


Drug Test. Analysis (2014) Copyright 2014 John Wiley & Sons, Ltd.
Drug Testing
and Analysis A. Sharma et al.

Table 2. Continued
S. No. Drug/ biomarker/ disease Application Analytical technique Reference
60. Lymphoproliferative diseases Detection of monoclonal immunoglobulin PCR
gene rearrangement
61. Phenylalanine and tyrosine Diagnosis of phenylketonuria Capillary electrophoresis-MS
62. Phenylketonuria Phenylalanine and tyrosine quantication LC-MS/MS
in neonatal blood
63. Phosphatidylethanol Monitoring of alcohol misuse LC-MS/MS
64. Pompe disease For diagnosis of Pompe disease Enzyme assay
65. Pregnancy- associated plasma First-trimester prenatal screening Enzyme assay
protein A and free -subunit of for aneuploidy
human chorionic gonadotropin
66. Prolactin For diagnosis of seizures Immunoenzymatic assay
67. Prostate specic antigen Prostate cancer screening Chemiluminescent Immunoassay
68. Prostate specic antigen (PSA) Mass screening for prostate cancer Chemiluminescent immunoassay
69. Retinol, tocopherols, beta-carotene, Malnutrition assay in HPLC
vitamin C and homocysteine developing countries
70. Somatomedin-c In diagnosis of pituitary gland RIA
disorder and abnormalities in
growth hormones production
71. S. pneumoniae and H. inuenzae b Dignosis of Pneumonia RT-PCR
72. Succinylacetone Diagnose of Tyrosinemia type 1 LC-MS/MS
73. Succinylacetone, amino acids New born screening for FIA-MS/MS
and acylcarnitines Tyrosinemia type I
74. Tay-sachs and sandhoff disease For detection of Tay-Sachs and MS/MS
Sandhoff disease
75. Thyroglobulin Thyroid function test Fluoroimmunometric Assay
76. Thyroid-antibody For detection of thyroid disease ELISA
77. Thyrotropin (TSH) and Thyroxine (T4) For diagnosis of congenital Fluorescent immunoassay
78. Thyroxine For diagnosis of hypothyroidism RIA
79. Thyroxine binding globulin Identication of levels changing of RIA
thyroid hormone
80. Transferrin Receptor Iron deciency Immunoassay
81. Triglycerides Diagnosis of heart diseases Enzyme assay
82. Triiodothyronine (T3) Diagnosis of congenital hypothyroidism ELISA
83. Thyroid stimulating hormone Diagnosis of congenital hypothyroidism ELISA
84. Vitamin D3-25-OH and In diagnosis of hypovitaminosis D LC-MS/MS
Vitamin D2-25-OH
85. 1-antitrypsin genotypes Early diagnosis of hereditary disorder, Fluorometric elastase [222]

associated with early risk of onset inhibition assay

chronic obstructive pulmonary disease
and liver dysfunction
86. -galactosidase Dignosis of Fabry disease Chip-based nanoelectrospray [223]

ionization mass spectrometry

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

of quality control samples with different hematocrit levels can in chromatographic retention time and peak shape distortion.
reduce the quantiable and unacceptable deviations. [1,32] Treated DBS cards consist of several types of proprietary reagents
Precautions are required in metabolomic proling using DBS as like sodium dodecayl sulfate, tris(hydroxymethyl)aminomethane,
some of the metabolites namely L-lysine, iminodiacetic acid, guanidinium thiocyanate, and others, which interrupts either in
DL-threo-beta-hydroxyaspartic acid, citric acid, adipamide, and ionization due to their non-volatile nature resulting in suppressed
adenosine-5-monophosphate, are found to be absent in DBS ionization or reduced signal intensity and often results in ion-
extracts but are detected in blood or plasma. [49] Another pitfall, paring with analytes leading to chromatographic peak shape
which is usually overlooked during analyte analysis from DBS deformity and altered retention time. This necessitates careful
cards through LC-MS/MS, is the potential interaction between evaluation of possible DBS card interactions and interferences.
analyte and card constituents which are sometimes culprits for Moreover, DBS method validation is more error prone
ionization suppression in the MS source and also for a change and time consuming in comparison to conventional plasma Copyright 2014 John Wiley & Sons, Ltd. Drug Test. Analysis (2014)
Drug Testing
An overview on dried blood spots and Analysis

samples as estimation of several supplementary validation References

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