Comparison between dried blood spot and conventional plasma analysis for the determination of the blood-to-plasma ratios of drugs with different plasma protein-binding properties

Comparison between dried blood spot and conventional plasma

analysis for the determination of the blood-to-plasma ratios of drugs

with different plasma protein-binding properties

Javier Antonio Mena Arenas

200410886

Thesis for the degree of Bachelor in Chemistry

Advisor: Roland J.W. Meesters, Ph.D.

Universidad de los Andes

Faculty of Science

Chemistry Department

2

List of abbreviations and acronyms

DBS Dried Blood Spots

DMPD Dried Matrix Paper Disks

LC Liquid chromatography

TOFMS Time of flight mass spectrometer

IS Internal Standard

Ht Hematocrit

HRMS High Resolution Mass Spectroscopy

IgG Immunoglobulin G

HDL High Density Lipoproteins

LDL Low Density Lipoproteins

3 Table of Contents

1.0 Introduction ... 7

1.1 Capillary and arterial blood ... 8

1.2 Blood traditional and microsampling techniques ... 9

1.2.1 Hematocrit effect and DMPD ... 10

1.3 Blood-to-plasma ratio ... 11

1.4 Protein binding of drugs ... 15

1.5 Acetaminophen... 18

1.6 High Resolution Mass Spectrometry (HRMS) ... 19

2.0 Objectives ... 22

2.1 General objective... 22

2.2 Specific objectives... 22

3.0 Methodology ... 22

3.1 Reagents and materials ... 22

3.2 HPLC instrumentation... 23

3.3 High Resolution Mass Spectrometry... 23

3.4 Blood sample collection ... 23

3.5 Sample preparation and ACP plasma analysis ... 24

3.6 ACP analysis applying the DBS microsampling technique ... 25

3.7 Hematocrit effect and DMPD analysis... 25

3.8 Determination of Ht value of whole blood... 26

3.9 Determinations of the blood-to-plasma ratio at different temperatures ... 27

4.0 Results ... 27

4.1 Linear regression analysis ... 27

4.2 Determination of blood-to-plasma ratios ... 28

4.3 Statistical comparison between methods... 29

5. Discussion ... 30

6. Conclusions ... 30

7. Acknowledgement ... 31

4

9.0 Appendices ... 35

9.1 Calibration curves ... 35

9.2 Calibration regression lines ... 41

5

Figures Index

Figure 1. Physicochemical composition of blood………8

Figure 2. Events occurring within blood………. 14

Figure 3. 3D-molecular structure of Albumin interacting……… 16

Figure 4. 3D molecular structure of the AAG protein……… 17

Figure 5. Low density lipoprotein interacting with lipid molecules……… 17

Figure 6. Conventional venous blood collection……….24

Figure 7. Preparation of DBS specimen………. 25

Figure 8. Preparation of DMPD specimen……….. 26

Figure 9. Determination of Ht of whole blood………27

6

Table Index

Table 1. Plasma protein binding of common drugs………...18

Table 2. Observed blood-to-plasma ratios of ACP at different Ht and temperatures………29

Table 3. Statistical results from testing the precision of both micro-sampling techniques.. 29

Table 4. Regression analysis results……….…..41

7

1.0

Introduction

Blood is one of the most biological matrixes analyzed in scientific research, especially in

biochemistry and in medical applications. Blood has a complex chemical composition; basically,

it is a suspension of different types of blood cells in an alkaline (pH ~ 7.0-8.0) and isotonic

aqueous medium known as plasma (1). Molecules like proteins, sugars, lipids, ions, gases,

hormones, electrolytes are also present in plasma. Blood cells include cells like Leukocytes,

platelets, erythrocytes, it is important to note that erythrocytes represent almost 99% of the blood

cells. Erythrocytes also known as red blood cells are responsible for the oxygen transport to

tissues and organs. The shape of this cell is essential for this function because they are biconcave

shaped and they have therefore a big surface area, which increases the efficiency of diffusion of

oxygen (O2) and carbon dioxyde (CO2) into and out of the cell.

Erythrocytes don’t have a nucleus and the major present molecule in the erythrocyte is the

hemoglobin molecule followed by membrane glycoproteins which determine the blood type of

individuals. The volumetric portion of the blood that is made up by erythrocytes is known as the

hematocrit. The hematocrit is thus defined as the volumetric ratio between erythrocytes and total

plasma. Hematocrit can vary; by gender, physical activities and environmental conditions. The

normal average hematocrit ranges between 40-50 % (males) and 38-45% (women) but this value

can be bigger in athletes (> 50%). The hematocrit value of blood rises if oxygen levels drop

down (higher altitude) or by doing aerobic exercises (rapid consumption of oxygen by skeletal

muscle) by stimulation of erythrocyte production in the bone narrow (erythropoiesis). When

hematocrit increases, the oxygen transport capability of blood is increases.

The liquid part of the blood is known as serum or plasma (serum is plasma plus coagulation

proteins like fibrinogen and other molecules). Plasma is a very important; it represents 55 % of

the blood volume and consists of 91 % of water, 7 % of proteins and 2% of nutrients. It is

important to know that ~58% of the in plasma present proteins are Albumin, 15% is IgG, besides

lipids carriers proteins such as Low Density Lipoprotein (LDL), Very Low Density Lipoprotein

and High Density Lipoprotein (HDL). The main functions of the blood proteins include: partially

regulation of blood’s pH, binding and transport of substances and molecules, immune system

functions (immunoglobulins), and blood coagulation (fibrinogen). But except proteins also

8

phosphate and organic acids, plasma contains cations predominantly sodium and lower presence

of potassium, calcium and magnesium are present in blood(1).

Image from: http://www.biosbcc.net/b100cardio/htm/blood.htm

Figure 1. Physicochemical composition of blood.

1.1

Capillary and arterial blood

The blood is distributed in the body by a system of vessels known as the circulatory system and

through them travel molecules and/or substances produced and/or introduced into the body.

There are three kinds of vessels; these vessels are different in size and localization into the body;

capillary, venous and arterial vessels. When we decide to make a chemical analysis using blood,

it is very important to select the correct type of blood; this due to concentration effects of certain

analytes that can occur in different blood vessel types.

Used methods to collect blood are: skin puncture (capillary blood) and venipuncture (venous

blood). Capillary blood obtained by skin puncture is done by puncturing fingertips, heels, toes

and earlobes. Capillary vessels connect the circulation of arterial and venous blood so that the

blood that flows through them is neither venous nor arterial; it has in principle a more arterial

than venous character, because when samples are collected, this blood can become

“contaminated” with interstitial and intracellular fluids depending on the pressure exerted around

9

between venous and capillary blood (2), in capillary blood for example it was found that in just

5% of the cases a higher albumin concentration than in venous blood, but in general the values

obtained from capillary and venous blood sampling are quite similar. Because of these

differences, we have to validate analytical methods using capillary blood first. Venous blood

samples are taken from the body by puncture of the anticubital vein, where also a tourniquet is

used in this procedure. It is known that prolonged use of a tourniquet can increase the

concentration values of no diffusible blood components.

1.2

Blood traditional and microsampling techniques

When blood is needed to be analyzed, the sampling of blood is an important parameter of the

analysis. The accuracy of analysis using blood samples can be seriously affected by improper

blood sampling techniques such as hemoconcentration due to excessive use of tourniquet, bad

puncture posture, hemolysis of samples and not optimal conditions of transport and storage.

Traditionally, the blood is collected by venipuncture, which is an invasive method, where a steel

needle is force-inserted into the anticubital vein and blood is collected in a plastic blood

collection tube containing anticoagulant. This method is being used for many years, the quantity

of blood collected is for example greater compared to capillary blood sampling. Moreover this

traditional method has some disadvantages; the storage and transport of big quantities, the way

of samples are preserved, it is a very invasive and difficult sampling method in especially

patients like children, chronic disease patients and the elderly.

In the early 1960’s a new blood collection method was introduced. One of its first applications

was the use of this so called dried blood spot method (DBS) to monitor inherited metabolic

disease such as Phenylketonuria (PKU) in the blood of newborns (3). Recently DBS has

experienced growing popularity because of it versatile and promising sampling technique in

clinical, medical, bioanalytical and pharmaceutical uses and applications. DBS applies the

collection of blood by finger prick or heel prick in combination with the use of blood in small

amounts (< 100 uL). The blood is collected onto cellulose filter paper, dried and target analytes

are extracted from the paper with an appropriate solvent and target analyte concentrations

determined by quantitative instrumental methods like liquid chromatography (LC) or gas

10

techniques. DBS is a microsampling technique that can offer a variety of advantages over the

traditional plasma, serum and whole blood collection: the blood samples can be collected in a

significantly smaller amount (~10-20 L); this makes DBS ideally suited for elderly, neonatal and infant screening and allows also a less invasive sample collection technique (4). Also DBS

sample storage (at room temperature) is easier, because in contrast to conventional liquid

sampling procedures, refrigeration and freezing is not necessary and can therefore be avoided, it

reduce shipping costs, reduces study animal numbers, and has many applications like screening

of metabolic disorders in children, diagnosis of viral infections like HIV and Hepatitis C (5) by

DNA and RNA polymerase chain reaction and it also reduce the infection risk due the samples

volume and quantitative bioanalysis (6). A unique attribute of the DBS sampling is that it allows

samples to be collected by patients themselves or guardians at the patient’s home or rest places

and samples can be posted by mail to laboratory for analysis, this provides an excellent clinical

monitoring method at any sampling time and results can be available at the clinic during any

routine checkup later (7).

1.2.1 Hematocrit effect and DMPD

One of the more considerable disadvantages using DBS are the presence of some sources of

error, like the paper characteristics, extraction procedures, analyte physicochemical properties,

not accurate spotting of the blood onto the paper, the spot position on the card, the punch

location and the Ht level of the blood sample, the latter is very critical this due to variation of the

Ht from 28-67% and will change based on gender, age, health and disease (6). The main effects

are the change of blood viscosity and diffusion rates, where the spot area is inversely

proportional to the Ht, so this affects the spot size which impacts the validity of the results

generated by DBS methods (8). To eliminate the Ht effect experimental approaches have been

developed in order to resolve and manage this effect. For example using DBS micro-sampling

applying whole-cut of the samples, this involves a complete punching of the DBS spots. Another

way to eliminate the Ht effect is by spotting blood onto pre-punched fixed size disks, this

controls the spot size at various Ht levels and results in DBS samples with the same blood spot

size. Actually there are two different whole-cut DBS microsampling techniques; one that uses

11

with the difference that the pre-cut techniques applies a support for the paper disks and the

perforated disc technique not. Both techniques have shown to minimize or completely eliminate

the Ht effect. In particular one of the best techniques to control the Ht effect in analysis is called

Dried Matrix on Paper Disks (DMPD)(9).

This microsampling technique applies the use of fixed size paper disks cartridges that improve

the efficiency of the blood spotting, storage, transport and shipping of samples. Other advantages

are the purchase of cartridges filled with precut desire papers inside that can be used at any time,

and easily can be spotted, extracted and analyzed. A minor drawback for the use of DMPD is the

precise volumetric blood sampling but this can be resolve using precise pipettes or glass

capillaries in the laboratory. The methodology of the DMPD technique is described in Section

3.7.

1.3

Blood-to-plasma ratio

In principle, administrated drugs are distributed in the blood stream as follows; a certain fraction

of the drug is bound to proteins, a second fraction is free available into the blood and the third

fraction is after a while intracellular available in erythrocytes and white blood cells. This

distribution principle can be used to determine total concentration of the drug in the whole blood,

and when the drug whole blood concentration is compared with the drug concentration obtained

from plasma analysis, we can estimate the bound and free non-bound drug fraction. This

distribution is called Blood- to-Plasma Ratio.

In general terms, when a drug is distributed between two phases (A and B), this distribution can

be determined by a partition coefficient PAB= CA/CB, where CA and CB are the concentrations of

the compound in each phase, so in principle this coefficient can be determined by measuring

both concentrations (10). Also the distribution coefficient can be determined by dissolving a

known amount of a study compound in an A-B system, which has different volume ratios of each

phase, so this permits to obtain PAB by measure of the concentration in one phase only.

With this principle we can determine the Blood-to-Plasma ratio, using two phases: plasma and

12

r= Cb/Cp(1)

Where Cb is the total drug concentration in blood and Cp is the total concentration in plasma.

To determine the value of r, one easy and efficient method is proposed, it consists of fortifying

the blood with the drug of interest. Then an aliquot of the fortified blood sample (known volume)

is transferred into a new tube, which contain a fixed volume of blank plasma. Then both fractions

are homogenized by mixing and thereafter centrifuged and the plasma supernatant is separated.

The concentration of drug in the plasma fraction is then determined by a quantitative analysis.

When the plasma drug concentration is obtained, we can derive the equation that allows us to

calculate the value of r.

The distribution of a compound in blood is characterized by its partition coefficient, between

blood cells and plasma,

p = Cbc/Cup = Cbc/Cpfup(2)

where Cbc is the drug concentration in blood cells, Cup is the unbound drug concentration in

plasma, Cp is the total drug concentration in plasma, and fup is the unbound fraction in plasma (fup

is considered constant and can be taken from literature or experimentally determined) .

The blood volume is composed mainly by the volume of blood cells Vbc = Vb and volume of

plasma Vp = Vb (1-Ht), where Vb is the total blood volume and H is the hematocrit (physiological

ranging from 0.4 to 0.6), so the concentration of drug in blood is

Cb = CbcVbc +CpVp/Vb = CbcHt + Cp (1-Ht) (3)

Then substituting (3) in (1) and using (2) we get the expression for Blood and plasma ratio

r= 1-H +pfupHt(4)

Considering the dilution of the blood sample with blank plasma, we define the dilution factor d

as

13

whereVbl is the volume of blood sample and Vadd is the volume of added plasma.

With this equation we are able to obtain the blood to plasma ratio comparing the drug

concentration in plasma in both undiluted and diluted blood samples.

So applying (1) and (5), we can calculate the concentration of compound in diluted blood, Cb,dil

Cb,dil= CbVbl/Vbl+Vadd = Cb/d = rCp/d (6)

In the same way the hematocrit in diluted samples is

Hdil= VblHt/Vbl + Vadd = Ht/d(7)

To obtain the blood to plasma ratio of the diluted blood sample we substitute (7) on (4)

rdil = 1-Hdil+pfupHdil=1-Ht/d+pfupHt/d (8)

Using(7) on (4) we can determine the concentration on plasma of the diluted blood sample Cp, dil

Cp,dil= Cb,dil/rdil = rCp/d-Ht+pfupH(9)

We can determine the ratio of the original and diluted plasma compound concentration with the

relation B

B = Cp/Cp, dil(10)

And using (4), (9) and (10) we obtain an equation to calculate r

r= d-1/B-1 (11)

When drugs enter into the blood, they interact with plasma proteins and red cells proteins and

14

Figure 2. Events occurring within blood; Cb: Bound concentration; Cu: Unbound concentration.Image taken from (10)

To understand the distribution of drugs in blood we need to consider these relations>

C=Cu/fu

Cb= [1-Ht/fu + Ht] Cu

r = Cb/C= (1-H) + Ht fu Where,

Cu = unbound concentration

C = plasma concentration

Cb = total blood concentration

fu = unbound fraction in plasma

Ht = hematocrit

= blood cell-to- unbound plasma concentration ratio

As we can see the total plasma and total blood concentration are proportional to Cu when fu,

and H are constant, in these situations both DBS and plasma analysis can be used to

determinate Cu. But when these conditions differ, we can evaluate such situations by the use of

the blood to plasma ratio. For example, some hydrophilic drugs like hydrophilic ones, therapy

15

erythrocytes (we need to take in account that blood cells of interest are only erythrocytes,

because the fractional volumes of white cells and platelets are so low as in general no to affect

the value of  (1)), in this situations blood cells simply act as diluent and the only influent on plasma binding is the hematocrit, if DBS is used. Other drugs such as caffeine and alcohol enter

into the erythrocytes but don’t bind with intracellular proteins and also don’t bind with plasma

proteins neither. In this case the total blood concentration is equal in cells and plasma and the

determinations can be done using DBS. In contrast some drugs bind to the cells and plasma

proteins; in this case we need to know what parameter fu, is the more important influencing the relation between Cu and total concentration.

Moreover, is important to validate and study DBS applications on toxicokinetcs and

pharmacokinetics studies, this implies the determination of the blood to plasma ratios, using this

microsampling technique, because DBS samples are in essence the same as whole blood and

have almost the same issues concerning interpretation of pharmacokinetics parameters. However

is important to establish if DBS is appropriate for some determinations. In drug discovery and

development, is useful to convert blood concentrations to unbound drug plasma concentrations,

and this can be done using blood to plasma ratio (only if it is constant). When the study matrix is

whole blood (like DBS analysis), we probably can determinate blood to plasma ratio, by using an

estimate drug plasma concentration by calculation of the concentration using the hematocrit

value of the used blood. For DBS analysis we can use the following relation(12):

R = 1+ (Cbc/Cp -1)* Ht (12)

where R is the B:P concentration ratio, Cbc is the blood cell drug concentration, Cp is plasma

drug concentration and Ht is the hematocrit.

1.4

Protein binding of drugs

As mentioned before, many drugs have interactions with proteins presented in plasma and body’s

tissues, also with other biomacromolecules (e.g. DNA), these interactions form

drug-macromolecule complex or adducts, in the case of plasma proteins (or others proteins) the

16

When irreversible the drugs bonds strongly to the proteins by a covalent chemical bonding, these

kinds of interactions are close related to inducing toxicity of certain compounds (13). For

example the hepatoxicity of Acetaminophen is due to presence of metabolite intermediaries that

interacts with liver proteins. The reversible process involves weaker chemical interactions as

hydrogen bonding or van der Waals forces due to presence of functional groups such as hydroxyl

or carboxyl in the amino acids of the proteins. This process is very important in

pharmacokinetics studies, because when the drug and the protein binds to form a molecular

complex it cannot transverse cells or capillary membranes, so this indicates that in the

drug-plasma protein bind, the compound is not biological active, but when it is free or unbound it can

crosses cells membranes and becomes active. The main active proteins found in plasma are (13):

- Albumin: Molecular weight of 65,000-69,000 Da, it’s produced in the liver and is the most

present reversible binding protein in the plasma, also is found in extracellular fluid of muscles

and skin. This protein is responsible of transport of endogenous and exogenous molecules such

as free fatty acids, hormones, tryptophan, weak acids make strong bonding with albumin

(e.g.salicylates, phenylbutazone, penicillins).

Figure 3. 3D-molecular structure of Albumin interacting with Naproxen (Image taken from

http://www.cambridgemedchemconsulting.com/resources/ADME/distribution.html)

- Alfa-1-acid glycoprotein (AAG): Globulin protein with molecular weight of 44,000 Da, its

17

antidepressants, neuroleptics and local anesthetics (e.g. propranolol, lydocaine) also transport

endogenous substances such as corticosteroids and the capacity is lower than albumin. This

protein is negatively charged at physiological pH.

Figure 4. 3D molecular structure of the AAG protein (Image taken from http://www.cambridgemedchemconsulting.com/resources/ADME/distribution.html)

-Lipoproteins: Complexes of lipids and proteins and are classified according the densities, this

molecules are known as VLDL (very low density lipoproteins), LDL (low density lipoproteins)

and HDL (high density lipoproteins). These proteins transport lipids to the liver across the

plasma and can transport exogenous compounds if the albumins get saturated.

Figure 5. Low density lipoprotein interacting with lipid molecules (Image taken from

18

The drug protein binding is determinate by some aspects such as: the physicochemical properties

and concentration of drugs in the body. The quantity of protein available for binding and the

physicochemical properties of it. The affinity between drug and protein and the interactions and

competition of the drug with other drugs and molecules, the health profile of patients and

individuals.

Table 1. Plasma protein binding of common drugs

Protein Drug

Albumin Ceftriaxone (A), Clindamycin (A), Clofibrate (A), Dexamethasone (N), Diazepam (N), Diazoxide (A), Dicloxacillin (N), Digitoxin (N),

Etoposide (N), Ibuprofen (A), Indomethacin (A), Nafcillin (A), Naproxen (A), Oxacillin (A), Phenylbutazone (A), Phenytoin (A), Probenecid (A), Salicylic add (A), Sulphisoxaole (A), Teniposide (N), Thiopental (A), Tolbutamide (A), Valproic acid (A), Warfarin (A).

AAG

Alprenolol (B), Carbamazepine (N), Disopyramide (B), Erythromycin (B), Lidocaine (B), Meperidine (B), Methadone (B), Verapamil (B).

Lipoproteins

Cyclosporine (N), Probucol (N).

A=Acid; B=Base; N=Neutral.

1.5

Acetaminophen

Acetaminophen (ACP), also known as Paracetamol and N-acetyl-p-aminophenol is a frequently

and widely used centrally acting analgesic and antypiretic drug in adults and children. It was

synthesized in the late 1800’s and Von Meringin was the first to report analgesic activity around

1893(14). This compound is sold around the world under various trade names, in a

mono-formulation and in addition with other compounds to control and regulate for example flu

19

pharmacokinetics of acetaminophen has been studied from neonates to elderly. It has been

proved that the compound is widely distributed in most of the body’s tissues and has a plasma

protein-binding rate of 10-25% at therapeutic concentrations. The recommended therapeutic

dosage for adults is 1000 mg per 100 mL of blood every 6 hours and for children (2-12 years

old) recommended dosage is 15mg/Kg every 6 hours (15). One negative aspect of the use of

ACP is its nephrotoxicity and hepatotoxicity related to the drug abuse, it was recognized until

1960 that overdose produce centrilobular liver necrosis, this refers to necrosis on liver central

tissue (16). For this the use of the compound has been restricted to patients with hepatic disease,

alcoholism, severe renal impairment and chronic malnutrition.

1.6 High Resolution Mass Spectrometry (HRMS)

Generally mass spectrometry is a widely used quantitative and qualitative analytical technique; it

studies and analyzes the masses of atoms, molecules or molecular fragments. To detect and

obtain de the mass spectrum of desired species, they are ionized when the gaseous species get

desorbed form the condensed phase. These ions are accelerated by an electric field and then

separated by their mass-to-charge ratio (m/z). This technique can be coupled to chromatography

instrumentation as an excellent quantitative and qualitative detector, because the mass

spectrometer can be high selective for the desire analyte.

One of the most used forms of the technique is the well-known High Resolution Mass

Spectrometry; the advantage of this technique is that an ion can be identified differentially from

other with same m/z, so what really does the spectrometer is distinguish very small differences in

mass, for example it can be resolve differences of 0.001 or less at m/z 100 (17). So the mass

resolution and mass accuracy increase. Some examples of HRMS instrumentation are:

double-focusing mass spectrometer, a time-of-flight mass spectrometer, Orbitrap mass spectrometer, or a

Fourier Transform Ion Cyclotron Resonance-mass spectrometer (FTICR-MS).

The time of flight mass spectrometry determine the m/z ratio using a time measurement.

Generally the ions are accelerated by an applied electric field and thus after acceleration the ions

get a kinetic energy depending on the charge. The ions need to travel until the detector over a

known distance taken some time for ions. This time depends also on the m/z ratio, if ions have

20

detector first. The best way of imagine this kind of spectrometer is imagine it as a long and

straight tube with the ionization source in one end and the detector in other, the ions that fly from

the source reach the detector in order of increasing mass, because lighter ones travel faster than

heavier (17). According to this travel time the m/z ratio of unknown can be determinate. Some

advantages of the use of the instrument are: high m/z accuracy, high acquisition rate (102 to 104

spectra/s) and also the measure of very high masses (m/z 106) (18).

1.7 Significance Tests

One of the most important requirements when an analytical method is applied or developed is

that it should be free from bias. Generally, this can be tested by measurement of quality control

samples with two different analytical methods, one method is mostly a “golden standard” method

and is seen as being analytical state-of-the art. In order to decide if there exists a statistical

difference between both methods and if errors can be accounted for as being random errors a

statistical tool known as a significance test can be used. This statistical test gives information on

potential statistical differences between the results obtained from both methods. For statistical

testing, all significance tests use a null hypothesis represented by H0 that states a statistical

hypothesis about both methods. In contrast, there is an alternative hypothesis represented by Ha

which describes the opposite statement of H0.

When the test has been applied we can conclude if the methods give statistically equal or

different results. So, H0 is rejected and Ha accepted or vice-versa.

The hypothesis are based in terms of population parameters, such as standard deviation (or mean (The hypothesis may be one sided (or one tailed) or two sided (two tailed) tested.)(19).

1.7.1 Fisher F-test for the comparison of standard deviations

In many cases it is important to make statistical comparisons between standard deviations of two

methods, i.e. the random errors of between the sets of data. This statistical analysis can be done

one-sided, when it is wanted to know if one method is more precise than other or two-sided when is wanted to know if the methods differ in their precision (better or less). If it is desire to test

21

whether two standard deviations are significantly different a two-sided test must be used. The

F-test uses the ratio of the two sample variances(20).

F = / and >



The null and alternative hypotheses for F-test are:

H0 : 

a : 

If the calculated value of F is greater than the critical F value then the null hypothesis is rejected.

The values of FCritical for P=0.05 for two-sided test are given in Appendix No. 9.4.

.

1.7.2 Outliers discrimination and Q-test

When an experiment is performed, usually in the set or sets of data some values appear to differ

from the others. These “different” values are known as outliers, and a discriminant test should be

done on the set of data in order to conclude if the suspect value has to be rejected or retained.

This statistical analysis is important because the obtained value for the mean and standard

deviation of a set of data depends on the observed data. One of the most used outliers test is the

Dixon’s Q-test. For small sets of data (3-7 measurements or values) the test indicates if a suspect

value should be rejected by comparing the difference between the value nearest to the suspect

one with the range of the set. Use the Q test for an outlier is to test H0: All measurements come

from the same population(21). The Qcalculated is calculated as follows:

Qcalculated= |suspect value – nearest value|/largest value – smallest value

The Qcritical values for P=0.05 for a two sided test are given in Appendix No.9.5. If the Qcalculated

22

2.0

OBJECTIVES

2.1

General objective

Compare two bioanalytical sampling methods for the determination of blood-to-plasma ratios of

drugs with different blood protein binding capabilities:

- Applying traditional whole blood and plasma analysis,

- Applying DBS microsampling technique analysis and estimation of plasma

concentrations,

- Applying DMPD to analyze and control the hematocrit effect on the determinations

2.2

Specific objectives

- Determinate the blood to plasma ratio using in-vitro plasma analysis considering dilution with

blank plasma and analysis by LC-HRMS.

- Determinate the blood to plasma ratio in whole blood by DBS technique coupled to LC-HRMS.

- Determinate the blood to plasma ratio of when possible the three model drugs with different

blood protein binding capacities by the two methods proposed.

- Compare the effects of Ht levels on blood-to-plasma ratio determination.

3.0

Methodology

3.1

Reagents and materials

Reagents used were: Acetaminophen (Sigma-Aldrich, USA), internal standard Acetaminophen

d-4 (Cerilliant, USA), Trifluoroacetic acid (Alfa–Aesar, USA), HPLC grade

methanol(Sigma-Aldrich, USA), HPLC grade Acetonitrile (Sigma-methanol(Sigma-Aldrich, USA), ultrapure water (18.2MΩ.cm)

produced by a Direct-Q® 3UV Milli-Q apparatus. Materials used were: Cellulose DBS paper

(Whatman FTA® DMPK cards fromGE health, USA), DMPD plastic cartridges( RockTown

Technology and Services, USA), Heraus 17 centrifuge (Thermo Scientific, Germany),

Haematocrit-Rotor determination kit (Thermo Scientific, Germany), temperature controlled bath

23

3.2

HPLC instrumentation

The HPLC system used consist of an Agilent 1260 Infinity LC provided with a G1312B binary

pump and G1367E auto sampler system. All separations were performed using the same

validated method as described in (22) the conditions were: Column Waters Atlantis T3 (2.1 x

100mm x 5μm) at 35°C (isothermally). The separation gradient used was: mobile phase A (100%

H2O) containing 0.1% formic acid (v/v) and mobile phase B was acetonitrile (100%). The

applied separation gradient was: 2% B from 0 to 0.5 min, 2 to 30% B from 0.5 to 2.5 min, 30 to

95% B from 2.5 to 2.6min, held at 95% B for 1.4min and followed by 2% B until 6.0 minutes

and a post time of 5 minutes for to next injection. Applied flow was 0.3 ml/min and sample

injection volume was 20μL.

3.3

High Resolution Mass Spectrometry

For detection an Agilent accurate mass QTOF-MS 6520 system and as chromatographic

separation was used (22). The equipment worked in positive electrospray ionization (ESI) for

detection and quantification of ACP and ACP-d4.

The operational settings were: gas temperature: 300°C (N2); nebulizer pressure: 48 psig;

capillary voltage (Vcap): 3500 Volts; drying gas: 8 L/min; fragmentor voltage: 135 Volts;

skimmer voltage: 60 Volts.

3.4

Blood sample collection

Whole blood and plasma samples were obtained from the Basics Medical Sciences Laboratory

(Faculty of Medicine, Universidad de los Andes). The venous blood samples were collected from

voluntary individuals in plastic collection tubes with anticoagulant (EDTA). For the preparation

of plasma, whole blood samples tubes were centrifuged at 2500 rpm for 5 minutes at 4°C, and

plasma was transferred into empty tubes or Eppendorf collection tubes of 1.5 mL. All the blood

to plasma ratio determination experiments were made in vitro, and all the whole blood and

plasma samples were storage at -20 °C. The procedure of venous blood sampling is shown in Fig.6.

24

Figure 6. Conventional venous blood collection

3.5

Sample preparation and ACP plasma analysis

The determination of the blood-to-plasma ratio was done following the method proposed in (10).

Initially fortified blood was prepared at a concentration level of 50 ppm Acetaminophen from a

500 ppm Acetaminophen stock solution made with ultrapure water, and waiting equilibration of

compound in blood. Then this solution was diluted twice with blank plasma, after shaking and

allow 20 minutes the compound equilibrate between plasma and red cells, both samples (whole

blood and diluted with plasma sample) were centrifuged five minutes at 2500 rpm and 4°C. Then

a 30 μL aliquot of supernatant plasma was deproteinized with 21 μL of a 10% trifluoroacetic

acid (TFA) plus a solution of ACP-d4 in methanol (5 ng/μL), then it was centrifuged again 15

minutes followed by centrifugation for 15 minutes at 4°C at 13300 rpm. Finally 30 μL of the

free-proteins supernatant plasma was diluted twice with ultrapure water and analyzed with

LC-QTOFMS instrument with the conditions explained above. The method was performed by

triplicate for each sample and a plasma calibration curve was constructed for each analysis, all

the calibrators were prepared as mentioned, making serial dilutions of ACP in plasma, with the

following concentrations 0.0, 1.0, 2.5, 5.0, 12.5, 25.0, 50.0 and 100.0 μg/mL.

All this procedure was repeated using different Ht levels: 24%, 47%, 56% at 20°C and Ht: 46%

25

3.6

ACP analysis applying the DBS microsampling technique

For preparation of DBS samples, the method described above was used, prepared was the 50

ppm Acetaminophen whole blood solution and diluted twice with blank plasma. After

equilibration between phases, 15µL of both whole blood and plasma diluted blood sample were

spotted on DBS paper and left air dried for two hours, then each spot was punched using a

punching tool, obtaining 3 mm diameter disks, then all the disks were extracted using 50 μL of

ACP-d4 methanol solution (1ng/μL), ultrasound bath was used for 15 minutes and then

centrifuged for 15 minutes to remove any cellulose(from paper), finally this samples were

analyzed with LC-QTOFMS instrument with the conditions explained above. This was made by

triplicate. The DBS calibration curve was made, making serial dilutions of ACP in whole blood,

spotting 15 µL on cellulose DBS paper and applying the method mentioned, the calibrators had

the following concentrations 0.0, 1.0, 2.5, 5.0, 12.5, 25.0, 50.0 and 100.0 μg/mL. This same

procedure was repeated using different Ht at different temperatures levels: 24%, 47%, 56% at

20°C and Ht: 46% at 37°C.

Figure 7. Preparation of DBS specimen

3.7

Hematocrit effect and DMPD analysis

As mentioned before, generally one of the most limitations of the DBS microsampling and

analysis is the Ht. This is controlled using DMPD technique and it was followed as proposed in

(9). Initially like in the plasma and DBS methodology samples were prepared at 50 ppm

26

disks were punched from DBS cellulose paper and placed into DMPD plastic cartridges and then

each disk was spotted with 8 μL of each blood sample (whole blood and plasma diluted blood).

The paper disks were let air dried for two hours and then extracted using 100 μL of ACP-d4

methanol solution (1ng/μL), ultrasound bath was used for 15 minutes and then centrifuged for 15

minutes to remove any cellulose(from paper), finally this samples were analyzed with

LC-HRMS instrument with the conditions explained above. All sample were prepared and analyzed

in triplicate. The DMPD calibration curve was made by applying serial dilutions of ACP in

whole blood, spotting 8 µL on paper disks and applying the method mentioned, the calibrators

had the following concentrations 0.0, 1.0, 2.5, 5.0, 12.5, 25.0, 50.0 and 100.0 μg/mL.

To study the effect of Ht in the blood-to-plasma ratio determinations, all the methodology

performed for Plasma, DBS and DMPD was repeated changing the Ht percentages, there were

24%, 47% (normal Ht), 56%.

For the preparation of 30% Ht blood sample 300 μL of red blood cells (from centrifuged blood)

were diluted with 700 μL of plasma. Using the same principle other Ht values were prepared.

Figure 8. Preparation of DMPD specimen

3.8

Determination of HCT value of whole blood

It was necessary to control the Ht value of blood samples which were artificially prepared by

mixing red blood cells and plasma. This was done in the laboratory by applying 75 mm glass

hematocrit capillary tubes (Drummond Hemato-Clad Mylar) that contained ammonium heparin

27

capillary tubes were at least for 75% of their volume filled with blood and centrifuged at 13300

rpm for 12 minutes. Then the Ht was determined by using a hematocrit scale as presented below

in figure 9

Figure 9. Determination of Ht of whole blood

3.9

Determinations of the blood-to-plasma ratio at different temperatures

One of the aims of these in- vitro experiments was to determine blood-to plasma ratio values at

different temperatures. For this purpose the same methodology used (section 3.5, 3.6 and 3.7)

was repeated using blood at 37°C in order to simulate normal body temperature. So for this,

fortified blood was temperature controlled in a water bath by placing a tube with sample blood as

shown in figure 10.

Figure 10. Incubation of ACP fortified whole blood at 37 ºC

4.0

Results

28

Linear regression analysis was applied by using the least square regression method. The

regression curves obtained for each method and sample matrix (plasma, DBS and DMPD) are

presented in Appendix 9.1. The regression equations and regression coefficients for all methods

are shown in Table 4 (Appendix 9.2). It is important to mark that the correlation coefficients (r)

obtained for each curve are close to one, this means that all calibration curves fit to a linear

model where there is a proportional relationship between concentration of ACP and ratio

between ACP and internal standard ACP-d4. Furthermore, regression coefficients (r2) were also close to 1 showing a good regression analysis.

4.2

Determination of blood-to-plasma ratios

The experiments done for the determination of blood-to-plasma ratios can be divided into four

parts:

i) The determination of blood-to-plasma ratios in vitro at 20°C with the normal average

Ht value (~45%) and dilution with blank plasma (2 fold).

ii) In-vitro at 20°C with Ht ~30% and dilution with blank plasma (2 fold).

iii) In-vitro at 20°C with Ht ~60% and dilution with blank plasma (2 fold),

iv) In-vitro at 37°C (body blood temperature) with normal Ht value (~45%) and dilution

with blank plasma (2 fold).

ACP concentrations were determined by using calibration curves prepared from ACP calibrators

with following concentrations 0.0, 1.0, 2.5, 5.0, 12.5, 25.0, 50.0 and 100.0 μg/mL. The

calibration curves were constructed by plotting the ACP concentration on the x-axis and the ratio

of the areas of ACP and internal standard ACP-d4 on the y-axis.

For the calculations of the blood to plasma it was used equation (12) using all the acetaminophen concentration found with interpolation using calibration curves. Obtained blood-to-plasma ratios

29

Table 2. Observed blood-to-plasma ratios of ACP at different Ht and temperatures

Hematocrit (%)

Temperature (°C)

Blood-to-plasma ratio (SD)

DBS DMPD

Non-diluted Diluted Non-diluted Diluted

24 18 0,99 (0,04) 0,96 (0,003) 1,04 (0,01) 0,92 (0,03)

47 18 1,21 (0,08) 1,10 (0,05) 1,12 (0,07) 1,12 (0,07)

56 18 0,99 (0,04) 0,98 (0,01) 1,17 (0,17) 1,14 (0,06)

47 37 1,27 (0,05) 1,06 (0,03) 1,25 (0,07) 1,13 (0,03)

4.3 Statistical comparison between methods

In order to compare statistically the results obtained from each assay it was considered to use a

Fisher F-test (two-tailed, P=0.05) to compare the precision of both micro-sampling techniques.

Compared where the results from the blood-to-plasma ratio values obtained using DBS and

DMPD at following conditions:

- Blood with 24, 47 Ht and 56 % Ht at 18°C.

- Blood with 47 % Ht at 18°C and 37°C.

In table 5 (Appendix 9.3) all standard deviations and variances obtained from these experiments

which were used in Fisher F-tests are presented.

Table 3. Statistical results from testing the precision of both micro-sampling techniques

Hematocrit (%)

Temperature (ºC)

Micro-sampling Technique

Statistical Comparisona

DBS-DBSb DMPD-DMPDb DBS-DMPDb DBS-DMPDb

ND-NDc ND-Dc NDc Dc

24 18 1.56 5.44 11.11 1.30

47 18 2.56 3.44 2.64 1.96

56 18 16.00 8.03 18.06 16.00

a

statistical comparison by Fisher F-test b

30

5.

Discussion

According to the results from the experiments where DBS and DMPD methods were compared

followed by a statistical Fisher F-test it was observed that both methods preformed statistically

equal (Table 3). In both methods the effect of dilution of the blood prior to the blood-to-plasma

measurement was studied and it was observed that dilution of with ACP fortified blood with

blank plasma did not have any significant impact on observed blood-to-plasma ratio (Table 2).

Moreover, the effect of performing the experiments at a different temperature of Ht value also

did not significantly affect the observed blood-to-plasma ratios (Table 2). All obtained results

showed that the precision of both micro-sampling techniques were equal. This was surprising

because it was assumed that the influence of the Ht causing an Ht-effect in the DBS assay would

be from statistical significance. In principle, this means that for the determination of ACP both

micro-sampling assays can be applied but probably not in general for other drugs. The protein

binding of ACP is low (10%) and this might be an explanation why both micro-sampling assays

perform equally. Moreover, when using these assays for other drugs it is quite possible that both

assays will perform statistically different. This needs to be proved by application of other drugs

with a different protein binding properties. Furthermore, with both assays observed

blood-to-plasma ratios were equal to the blood-to-blood-to-plasma ratio reported in the literature (23), where a

blood-to-plasma ratio value of 1.1 was reported.

6.

Conclusions

It was concluded from the obtained results that it was possible to determine the blood-to-plasma

ratio of ACP using the new generation micro-sampling assays. Both assays demonstrated the

same precision and the precision was not depending on the temperature, Ht and if the

experiments were done by using blank plasma diluted or non-diluted whole blood which was

fortified with ACP.

The precision between both assays was equal but also the accuracy of both assays was good

since the observed mean blood-to-plasma ratio for ACP for DBS and DMPD were both 1.1. and

31

In this thesis it was proven that DBS and DMPD microsampling techniques are excellent tools

for the determination of blood-to-plasma ratios of drugs and these values are important in drug

development studies as well in pharmacokinetic studies. Moreover, the versatility of these

microsampling techniques needs to be tested with other types of drugs to see if certain properties

of drugs such as protein-binding to blood proteins and lipophilicty of the drug has an impact on

the precision and accuracy of these microsampling techniques.

7.

Acknowledgement

This study was supported and sponsored by Grupo de Investigación en Química Analítica y

Bioanalítica (GABIO) of Universidad de los Andes, Department of Chemistry, Bogotá D.C.,

Colombia and its director Dr. Roland J.W. Meesters. The author of this work would thanks to

Director and members of GABIO for all the support through the experimentation, also thanks to

The Basics Medical Sciences Laboratory of the faculty of Medicine from Universidad de los

Andes, Bogotá D.C., Colombia for the support and help in blood samples collection. Thanks to

all voluntary personal for blood provide and Mr. Edwin Guevara for the technical support and

LC-HRMS analysis.

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35

9.0

Appendices

41

9.2

Calibration regression lines

Table 4. Regression analysis results

Matrix Line equation Slope Correlation

coefficient (r)

Regression coefficient (r2) Plasma Ht 47% at

18°C

0,3681x 0,3681± 0,0060 0,9994 0,9978

DBS Ht 47% at

18°C

0,0193x 0,0193± 0,0047 0,9977 0,9988

DMPD Ht 47% at

18°C

0,0441x 0,0441±0,0113 0,9980 0,9945

Plasma Ht 56% at

18°C

0,7279x 0,7279±0,0459 0,9982 0,9959

DBS Ht 56% at

18°C

0,0582x 0,0582±0,0056 0,9952 0,9906

DMPD Ht 56% at

18°C

0,1759x 0,1759±0,0155 0,9972 0,9926

Plasma Ht 24% at

18°C

0,4268x 0,4268± 0,0669 0,9626 0,9988

DBS Ht 24% at

18°C

0,0556x 0,0556±0,0181 0,9042 0,9932

DMPD Ht 24% at

18°C

0,1284x 0,1284± 0,0184 0,9991 0,9927

Plasma Ht 46% at

37°C

0,1756x 0,1756±0,0019 0,9967 0,9906

DBS Ht 46% at

37°C

0,0592x 0,0596±0,1346 0,9996 0,9993

DMPD Ht 46% at

37°C

42

9.3 Statistical test

Table 5. Observed variances from experiments on the blood-to-plasma experiments

Hematocrit (%)

Temperature (°C)

standard deviation (S and Variance (S

DBS DMPD

Non-diluted S S

Diluted S S

Non-diluted

S S

Diluted

S S

24 18 0,04 0,0016 0,032 0,001024 0,012 0,000144 0,028 0,000784

47 18 0,08 0,0064 0,05 0,0025 0,13 0,0169 0,07 0,0049

56 18 0,04 0,0016 0,01 0,00015 0,17 0,0289 0,06 0,0036

43

9.4. Critical values of Fisher F for a two sided test (P=0.05)

44

9.5. Critical values of Dixon’s Q for a two sided test at various confidence

levels