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GHENT UNIVERSITY

FACULTY OF PHARMACEUTICAL SCIENCES

Department of Pharmaceutical Analysis

Laboratory for Drug Quality and Registration (DruQuaR)

Academic year 2012-2013

API LOSS DURING CAPSULE PREPARATION

Eline GRAULS

First Master of Pharmaceutical Care

Promotor

Prof. Bart De Spiegeleer

Commissioners

Prof. J.P. Remon

Prof. H. Robays

COPYRIGHT

"The author and the promoters give the authorization to consult and to copy parts of this thesis

for personal use only. Any other use is limited by the laws of copyright, especially concerning

the obligation to refer to the source whenever results from this thesis are cited."

May 22, 2013

Promoter Author

Prof. dr. Bart De Spiegeleer Eline Grauls

SUMMARY

Due to sub-label claim API content problems in capsules, this research investigated the

API loss during the preparation of capsules. This thesis consists of two parts. First, the

experimental part, where the pharmaceutical compounding of three molecules

(hydrocortisone, triamcinolone and dexamethasone) are evaluated on potential API losses.

Furthermore, the influence of some variables (e.g. mortar) are investigated in more detail. The

second part includes the datamining of a list of 17 molecules, in which the median assay

results of each molecule are modeled in function of the molecule structural descriptors,

examining a molecule effect on the observed sub-label claim API contents in capsules.

Our findings show that for the three molecules, the highest API loss occurred at the

mortar step. Therefore, the influence of this variable was investigated separately, from which

two main conclusions were drawn:

(1) the type of material of the mortar has an influence on API loss, i.e. an agate

mortar has a lower API loss than a porcelain mortar.

(2) the used state of the mortar plays an important role as well, i.e. a new mortar (not

used) showed a lower API loss than an old mortar (extensively used). The older/more

used/rougher the mortar becomes, the higher the API loss will be.

Another important result is that there is no loss of API at the capsule shell, which is

interesting for the pediatric/geriatric use.

The presence of a molecule (chemical) effect on the median assay values is demonstrated

based upon the results of the datamining. This molecule effect was modeled, by expressing

the 17 median assay values in function of corresponding molecular descriptors, using stepwise

MLR and PCA techniques. The obtained MLR model, containing 3 variables (RDF060m,

R6e+ and R3m

+) explained 92.5% of the observed % API loss variability, whereas the PCA

model explained 95%. The larger the variability in API loss is, the more sensitive the

compound is for operational variables (i.e., mortar type and condition, API concentration, no.

of capsules produced, personnel and capsulation device).

Based on our findings, future research efforts are needed to improve the API loss during

capsule preparation, i.e. to decrease the loss as well as its variability.

SAMENVATTING

Vanwege vaak voorkomende API problemen in capsules, analyseerde dit onderzoek het

API verlies tijdens de capsule bereiding. Deze thesis bestaat uit twee luiken. Het eerste luik is

het experimenteel gedeelte, waarin drie moleculen (hydrocortisone, triamcinolone en

dexamethasone) onderzocht worden op potentieel API verlies tijdens de bereiding. Bovendien

wordt de invloed van sommige variabelen (vb. mortier) nader onderzocht. Het tweede luik

bestaat uit datamining van een lijst van 17 moleculen, waarbij de analyse resultaten (mediaan)

van elke molecule worden gemodelleerd in functie van de structurele descriptoren van de

moleculen. Hier zal het molecule-effect op de waargenomen sub-label claim API inhoud in de

capsules onderzocht worden.

Onze bevindingen tonen aan dat voor de drie moleculen, het hoogste API verlies optrad

in de mortier stap. Daarom werd de invloed van deze variabele apart onderzocht, waaruit twee

belangrijke conclusies werden getrokken:

(1) het materiaal van de mortier speelt een belangrijke rol in het API verlies, d.w.z.

een agaat mortier heeft een lager API verlies dan een porseleinen mortier.

(2) alsook de toestand/conditie van de mortier speelt een belangrijke rol, een nieuwe

mortier (nog niet gebruikt) toonde een lager API verlies aan dan een oude mortier (veel

gebruikt). Hoe ouder/meer gebruikt/ruwer de mortier, hoe hoger het API verlies zal zijn.

Een ander belangrijk resultaat is dat er geen verlies van API aan de capsulewand optrad,

wat interessant is voor pediatrisch/geriatrisch gebruik.

Het deel datamining, toont de aanwezigheid van een molecuul (chemisch) effect op de

mediaan analyse waarden aan. Dit molecuul effect werd gemodelleerd met behulp van

stapsgewijze MLR en PCA technieken. Het verkregen MLR-model verklaarde 92,5% van het

geobserveerde % API verlies, terwijl het PCA-model 95% verklaarde. Hoe groter de

variabiliteit in API verlies, hoe gevoeliger het component is voor operationele variabelen

(d.w.z. type en staat van de mortier, de API concentratie, het aantal gemaakte capsules, het

personeel en de gelulier).

Op basis van onze bevindingen zijn toekomstige onderzoeksinspanningen nodig om het

API verlies tijdens de capsulebereiding te verbeteren, d.w.z. het verlagen van het verlies

alsook zijn variabiliteit.

WORD OF THANKS

“Many a little makes a mickle.”

(William Camden, 1614)

First of all, I want to thank my promoter Prof. Bart De Spiegeleer for giving me the

opportunity to perform this thesis and for his good advice.

Subsequently I would like to thank Apr. Matthias D’Hondt for his excellent guidance,

corrections and comments on writing this thesis.

I would also like to thank Prof. Jean-Paul Remon and Prof. Chris Vervaet for letting me use

the materials that I needed for this thesis.

I would like to thank everyone of DruQuaR as well for their help and pleasant atmosphere.

And finally I would like to thank my family and friends for their support during this thesis.

TABLE OF CONTENTS

1. INTRODUCTION .............................................................................................................. 1

1.1 GENERAL ................................................................................................................... 1

1.1.1 Drugs .................................................................................................................... 1

1.1.2 Routes of administration ...................................................................................... 1

1.1.3 Dosage forms ........................................................................................................ 2

1.1.4 Capsules ............................................................................................................... 3

1.2 CAPSULE PREPARATION ....................................................................................... 4

1.2.1 Industrial ............................................................................................................... 4

1.2.2 Pharmaceutical compounding .............................................................................. 4

1.2.2.1 Capsules ........................................................................................................ 5

1.2.2.2 Trituration ..................................................................................................... 5

1.3 API LOSS DURING MAGISTRAL CAPSULE PREPARATION ............................ 6

1.3.1 Problem ................................................................................................................ 6

1.3.2 Powder .................................................................................................................. 6

1.3.2.1 Mixing of powders ........................................................................................ 6

1.3.2.2 Physicochemical properties........................................................................... 7

1.3.2.3 Sampling ....................................................................................................... 7

1.3.3 Roughness degree of the surface .......................................................................... 7

1.3.4 Compounds ........................................................................................................... 7

1.4 ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY .................................. 8

2. OBJECTIVE ..................................................................................................................... 10

3. MATERIALS AND METHODS ...................................................................................... 11

3.1 CHEMICALS ............................................................................................................ 11

3.2 MATERIALS AND EQUIPMENTS ........................................................................ 11

3.3 METHODS ................................................................................................................ 12

3.3.1 UPLC–method and verification ......................................................................... 12

3.4 HYDROCORTISONE CAPSULE PREPARATION ............................................... 12

3.4.1 Hydrocortisone 10 mg capsules ......................................................................... 12

3.4.1.1 Capsule preparation .................................................................................... 12

3.4.1.2 Swabbing of working area and equipment + swab sample preparation ...... 12

3.4.1.3 Preparation of reference solutions............................................................... 13

3.4.1.4 Capsule sample preparation ........................................................................ 13

3.4.1.5 Residual capsule sample preparation .......................................................... 13


3.4.3 Mortar influence during hydrocortisone preparation ......................................... 14

3.4.3.1 Different mortars ......................................................................................... 14

3.4.3.2 Capsule preparation .................................................................................... 14



3.5 TRIAMCINOLONE CAPSULE PREPARATION: 4 MG ....................................... 15

3.6 DEXAMETHASONE CAPSULE PREPARATION: 0.25 MG ................................ 15

3.6.1 Influence of sample solvent and sample solvent strength .................................. 15

3.6.2 Preparation of own-made 10%-dexamethasone trituration ................................ 16

3.6.2.1 Protocol 10%-dexamethsanone trituration .................................................. 16



3.6.3 Analytical development and pilot recovery experiments ................................... 17

3.6.3.1 Filter and centrifuge influence .................................................................... 18

3.6.3.2 Influence mixing step only .......................................................................... 18

3.6.3.3 Influence swabbing technique ..................................................................... 19

3.6.4 Dexamethasone 0.25 mg capsules ...................................................................... 20

3.6.4.1 Protocol capsule preparation ....................................................................... 20



3.6.4.4 Capsule sample preparation ........................................................................ 21

3.6.4.5 Residual capsule sample preparation .......................................................... 21

3.7 DATAMINING ......................................................................................................... 21

4. RESULTS AND DISCUSSION ....................................................................................... 23

4.1 METHOD VERIFICATION ..................................................................................... 23

4.2 HYDROCORTISONE ............................................................................................... 23


4.2.1.1 % label claim capsules ................................................................................ 23

4.2.1.2 API mass balance ........................................................................................ 24



4.2.2.2 API mass balance ........................................................................................ 25

4.2.2.3 Comparison of hydrocortisone concentration in bulk and residual powder 26

4.2.3 Concentration effect ........................................................................................... 27

4.2.4 Mortar influence ................................................................................................. 28

4.2.5 Suggestion for improvement of hydrocortisone capsule preparations ............... 30

4.3 TRIAMCINOLONE .................................................................................................. 31

4.3.1 Triamcinolone 4 mg capsules ............................................................................. 31


4.3.1.2 API mass balance ........................................................................................ 32

4.4 DEXAMETHASONE ............................................................................................... 33

4.4.1 Own-made 10%-dexamethasone trituration ....................................................... 33

4.4.1.1 Dexamethasone content in own-made trituration ....................................... 33

4.4.1.2 Swabs dexametasone content and % API mass balance ............................. 33

4.4.2 Analytical development and pilot recovery experiments ................................... 34

4.4.2.1 Sample solvent strength influence .............................................................. 34

4.4.2.2 Filter and centrifuge influence .................................................................... 34

4.4.2.3 Influence swabbing technique ..................................................................... 35

4.4.2.4 Microscopy ................................................................................................. 35

4.4.2.5 Process: influence mixing step .................................................................... 36

4.4.3 Dexamethasone 0.25 mg capsules using 10% own-made trituration ................. 37

4.4.3.1 % label claim capsules (own-made trituration)........................................... 37

4.4.3.2 API mass balance (own-made trituration) .................................................. 38

4.4.4 Dexamethasone 0.25 mg capsules using a 10% commercial trituration ............ 39

4.4.4.1 % label claim capsules (commercial trituration)......................................... 39

4.4.4.2 API mass balance (commercial trituration) ................................................ 39

4.4.5 Comparison of the own-made and commercial trituration ................................. 40

4.5 INFLUENCE OF CHEMICAL STRUCTURE (DATAMINING) ........................... 41

4.5.1 Data .................................................................................................................... 41

4.5.2 Modeling ............................................................................................................ 42

4.5.3 Stepwise multiple linear regression .................................................................... 42

4.5.3.1 Model 1: All selected descriptors (n=1616) ................................................ 42

4.5.3.2 Model 2: selected descriptor groups (n=355) ............................................. 43

4.5.3.3 Model 3: selected descriptors (n=55) .......................................................... 43

4.5.4 Principal component analysis (PCA) ................................................................. 44

4.5.5 Discussion of models ......................................................................................... 44

4.6 FUTURE PERSPECTIVES ...................................................................................... 46

5. CONCLUSION ................................................................................................................. 47

6. REFERENCES ................................................................................................................. 48

7. ATTACHMENT ...................................................................................................................

8. EVENING LECTURES: INTERNATIONALISATION AT HOME ..................................

LIST WITH ABBREVATIONS

ACN Acetonitrile

ANOVA Analysis of variance

API Active pharmaceutical ingredient

DEX Dexamethasone

HC Hydrocortisone

HETP Height equivalent of a theoretical plate

HPLC High performance liquid chromatography

HPMC Hydroxypropyl methylcellulose

LC Label claim

LOD Limit of detection

LOQ Limit of quantification

MeOH Methanol

MLR Multiple linear regression

PCA Principal component analysis

PDA Photodiode array

PLS Partial least squares

QC Quality control

RSD Relative standard deviation

TMF Therapeutisch magistraal formularium

TRI Triamcinolone

UPLC Ultra performed liquid chromatography

UV Ultraviolet

1

1. INTRODUCTION

1.1 GENERAL

1.1.1 Drugs

Medicines evolved from natural products (such as plants or plants components) to

chemically synthesized drugs, in which the 19th

century was a breakthrough for the

pharmaceutical industry. A drug is defined as “a single or combination of substances with

therapeutic or prophylactic properties in relation to diseases in humans or animals”, according

to the Royal decree of 25/3/1964 [1]. The two main goals of drug administration are to improve

the patient’s health and the quality of life. Despite having positive, desired effects (therapeutic

effects), drugs can also carry negative, undesired effects (side effects), whereby these side

effects are often dose-related [2].

The role of the pharmacist has evolved significantly over the years, in which

pharmaceutical care has become more and more important, surpassing drug preparation. In the

field of pharmaceutical care, the pharmacist gives advice and feedback to the patient, doctor

and nurse, and is regarded as the expert of medicines. However, the role of the pharmacist still

comprises the preparation of medicines and the delivery of commercial preparations.

1.1.2 Routes of administration

Pharmaceutical drugs have a wide variety in properties and action mechanisms, resulting in

different administration routes and dosage forms, so each illness can be treated as optimal as

possible. The three main administration routes are given in Table 1.1 [3].

Each route of administration has his own advantages, and disadvantages and combined

with the specific drug properties and general patient preferences, an optimal administration

route can be chosen. The oral route is preferred by the most people, as this is a painless, safe,

easy and cheap administration route [4-6].

2

Table 1.1 Drug routes of administration

Route of administration Effect Form

Topical Local effect Skin / Mucosal membranes

Enteral Systemic effect (digestive tract)

Oral

Rectal

Buccal

Sublingual

Parenteral Systemic effect

Intravenous

Intramuscular

Subcutane

Topical administration means that the drug will be placed directly in contact with the skin

or the mucosal membranes. Enteral administered drugs will be absorbed in the bloodstream

from the gastrointestinal tract. The oral route is by mouth, the rectal route is along the rectum

(the last part of the colon), the buccal route is through the cheek and sublingual administered

drugs are absorbed beneath the tongue. Parenteral administration means that the drug is directly

placed into the bloodstream, which is mostly done through injections or infusions. Different

possible injection sites include under the skin (subcutane), in the muscle (intramuscular) and in

the vein (intravenous). Infusions are only given intravenous [7].

A local effect means that the drug is directly applied at the desired site of action, contrary

to the systemic effect in which the drug is absorbed in the bloodstream and transported to the

site of action. It is important to know that when a drug has a local effect, the drug can have a

systemic effect as well [7].

1.1.3 Dosage forms

Classically, three distinct dosage form types exist. The first is the solid dosage form, which

comprises powders, capsules, tablets, suppository and ovules. The second is the semi-solid

dosage form, which contains salves, creams, gels and pastes. And the third is the liquid dosage

form, which covers suspensions, emulsions, solutions and syrups. Each drug, can be processed

into different dosage forms, depending on its application or target public, e.g. children vs.

adults.

3

For each route of administration, a number of dosage forms exist. For example the enteral

route can be accessed through capsules, tablets and liquid formulations while the topical route

can be used through salves, gels, creams and pastes.

1.1.4 Capsules

Capsules are small containers which are filled with one or more active pharmaceutical

ingredient (API) and excipients such as filling agents, flowing agents, solubilizing agents and

antimicrobial preservatives [8]. The excipients need to be API-compatible, concerning

chemical stability, density, particle size and shape [9]. The capsule is filled with a drug dose,

which can be a solid, a semi-solid or a liquid formulation. Capsules are mostly gelatin-based,

which were invented in the 19th

century, primarily to mask the bad taste and unappetizing color

of the active substances [10]. Gelatin is prepared from collagen derived from the bones and the

skin of animals through partial hydrolysis. An alternative for gelatin is hydroxypropyl

methylcellulose (HPMC), which is an synthetic derivative of cellulose. The primary advantage

of HPMC is that it is derived from a vegetable source, which is important for vegetarians and

certain religions. Also, HPMC is compatible with hygroscopic products as HPMC itself has a

relatively low water content of 2-5%, while gelatin has a water content of 13-16% [11, 12].

Capsules are a popular dosage form because of their stability, the high dosage accuracy, quick

drug release and ease of swallowing.

There are two kinds of capsules, the hard gelatin capsules (exist out of two parts) and the

soft gelatin capsules (exist out of one part). The soft gelatin capsules, mostly filled with liquids,

exist in different sizes and shapes and can only be made in the industry [10].

In the pharmacy, hard gelatin capsules-based drug formulations can be made. The hard

gelatin capsules are mostly filled with a solid formulation and have a Coni-Snap® system for

secure closure to ensure that the capsules cannot be opened accidentally and/or unobserved.

Due to the Coni-Snap® system, a drama such as the “Chicago Tylenol murder” can be

prevented. Here, an unknown person poisoned Tylenol capsules (API:paracetamol) with

cyanide, which resulted in the death of seven people. Because of this incident, safe drug

packaging such as the Coni-Snap® capsules, which hampers tampering, are important to

guarantee the safety of the patients [13]. However, if a patient needs to open the capsule, the

capsules may not be locked into the Coni-Snap® capsule system.

4

Hard gelatin capsules exist in different sizes, ranging from n° 5 (smallest) to n° 000

(largest). The capsule sizes and their corresponding total volumes according to the

Therapeutisch magistraal formularium (TMF)” are given in Table 1.2 [9].

Table 1.2 Volumes of hard gelatin capsules in the pharmacy according to TMF

Capsule size

N° 3 2 1 0 00 000

Volume (ml) 0.27 0.37 0.48 0.67 0.95 1.36

According to the European Pharmacopeia, i.e. in the Dosage Forms monograph “Capsules”

(Ph. Eur. 1/2008:0016) and Pharmaceutical Preparations (Ph. Eur. 04/2013:2619), quality is

required [8]. There are three major aspects: (i) average weight/content, (ii) weight/content

distribution, (iii) dissolution and disintegration. These are specifications which can be tested by

quality control (QC).

1.2 CAPSULE PREPARATION

1.2.1 Industrial

Today, the majority of all drugs formulations, are prepared industrially [14]. The reason for

this increase of industrially produced medicines are the low costs, the high production speed,

the complexity of the new drugs formulations and the high quality requirements. A

disadvantage of the mechanical preparations of drugs is that the pharmaceutical industry

doesn’t allow or at least limits individualized medication, resulting in a “one size fits all”-

strategy [15].

1.2.2 Pharmaceutical compounding

Pharmaceutical compounding is the preparation of drugs formulations performed by the

pharmacist, which continues to play an important role in today’s health care system as it allows

for a more individualized, i.e. patient-specific, medication program [14]. It is important that

pharmacy students still possess adequate skills for pharmaceutical compounding, so a safe and

high quality drug product can be delivered to the patient [16].

5

1.2.2.1 Capsules

For certain pharmacy-made capsule preparations, TMF stipulates specific guidelines. TMF

is a reference manual where compounding methods are described as well as providing shelf

lifes and indications. Several materials are needed for capsule preparation. A weighing device,

needed for weighing API starting material, a mortar, pestle and flexible plastic card for mixing

the different starting materials (APIs and excipients) into one homogenous powder, a

measuring cylinder for obtaining a sufficient powder volume (API and excipients) to fill the

capsules, a paper funnel to transfer the powder from the mortar to the measuring cylinder and a

capsulation device (or gelulier) in which the powder mixture is filled out into the capsules [9].

A example of the mortar-pestle-flexible plastic card and capsulation device are given in Figure

1.1.

Figure 1.1: Mortar, pestle and flexible plastic card (left) and capsulation device (right)

1.2.2.2 Trituration

In the pharmacy, the minimum API weight which can still be measured accurately is set at

50 mg. If a lower API quantity is needed, a trituration (i.e. dilution) must be made, to ensure

sufficient accurate weighing. Excipients, containing diluents (e.g. lactose, mannitol, starch) and

lubricants (e.g. talc, colloidal siliciumdioxide, magnesium stearaat), as well as a small amount

of permitted colorant (color agent iron red, color agent riboflavine) are used in a trituration of

API [17]. The colorant enables visual confirmation of a homogeneous powder mixture. From

this trituration, a desired quantity will be taken corresponding to the desired API amount.

Beside pharmacy-made triturations, commercially available triturations can also be bought,

having i.a. the advantage of the availability of an analysis certificate confirming the quality of

the trituration [9].

6

1.3 API LOSS DURING MAGISTRAL CAPSULE PREPARATION

1.3.1 Problem

There has been several problems (mainly variability and content) with the API content in

pharmaceutical compounded capsules. Basically, the API content in capsules is often too low.

In 2011, an article was published in Testaankoop where this quality problem was cited. The

investigation of Testaankoop verified the preparation of 40 dexamethasone (0.5 mg) capsules

by 42 different pharmacists. Two pharmacists did not take the prescription, because they did

not possess the raw API material. The samples were checked against four requirements: (i)

weight distribution, (ii) average content and (iii) content distribution. Only 11 of the 40

samples, or approximately 27.50%, passed all these tests. Most disapproved samples had an

average API content outside of the 90-110% interval, whereas the weight distribution was

found to be fairly ok (32 of the 40 samples, or approximately 80%, passed this requirement).

Finally, (iv) labeling of the preparation was also checked on the required statements and only

one pharmacist was found to conform with the regulatory requirements [14].

As a direct result of previous API content studies, a ringtest (number 4) was performed, in

which the preparation of different capsules preparations, focusing on different APIs, was

investigated. As a result of this study, which confirmed the aforementioned sub-label claim API

content problem, a closer look on the capsule preparation process was required. This thesis will

examine the different steps included in the pharmaceutical capsule preparation process, and

evaluate the API losses in this process.

1.3.2 Powder

1.3.2.1 Mixing of powders

Mixing of powders is a technique which is used in many industries, including the

pharmaceutical industry. Here blending of particles, a technique to obtain a

homogenous/uniform mixture, is a commonly used step in the preparation of solid mixtures

such as tablets and capsules. For the blending of powders, several methods are used, ranging

from manual mixing with mortar and pestle to mechanical mixing. The mixing process is

influenced by the powders characteristics [18, 19].

7

1.3.2.2 Physicochemical properties

The physical and chemical properties of a powder are important to understand their

behavior. There are 2 types of powders, free flowing and cohesive powders. The particles in

free flowing powders do not adhere with each other, whereas particles in cohesive powders

have a relatively strong cohesion force. Cohesion is a force of attraction between equal

molecules [20]. The smaller the particle becomes, the more important the cohesion force is due

to the presence of the interparticle forces (e.g. Van der Waals Forces, dipole bonds, hydrogen

bonds, polarizability, aromaticity, electrostatic forces, hydrophobicity) [21].

1.3.2.3 Sampling

For analyzing a powder mixtures, samples should be taken (i.e. sampled). Various

materials can be used for sampling in pharmaceutical analyses (e.g. end-sampling probes).

Sampling is mostly done in triplicate and are preferably taken at different places in the powder

bed, so the samples are representative for the total powder mixture. The size of the sample is

often one to three times the final dosage unit. Sampling is an in-process technique. [21].

1.3.3 Roughness degree of the surface

During the capsule preparation, the powder mixture comes into contact with different

surfaces, e.g. mortar and/or the surface of the gelulier. The roughness of these surfaces will

also influence a potential powder and API loss. Generally, the possibility of any powder loss

increases with the roughness of the surfaces it comes into contact with. The mortar material ,

e.g. agate, melamin, porcelain, and gelulier material (plastic and metal) as well as the state of

use will influence the roughness of the corresponding surfaces.

1.3.4 Compounds

In Ringtest 4 following API’s were investigated by DruQuaR: hydrocortisone,

triamcinolone and dexamethasone. The molecular structure of hydrocortisone, triamcinolone

and dexamethasone is given in Figure 1.2.

8

OH

OH

CH3

H

O

CH3

H

H

OH

O

Hydrocortisone

OH

H

H

F

OH

CH3

CH3

OH

OH

O

O

TriamcinoloneO

OHO

CH3

CH3

CH3

OH

F H

H

OH

Dexamethasone

Figure 1.2: Molecular structures of hydrocortisone, triamcinolone and dexamethasone

These three molecules are corticosteroids, more specific glucocorticoids [22]. They are

therapeutically used in the therapy of inflammatory diseases and in immunosuppression [23].

Hydrocortisone, also named cortisol, is a natural glucocorticoid in humans. Triamcinolone and

dexamethasone are synthetic glucocorticoids, which have an analogue structure to

hydrocortisone but have a weaker mineralocorticoid effect and stronger glucocorticoid effect

[9, 22].

API content data of other molecules are also available and allow investigation of a

molecule-specific influence on the final API content in the capsule (see section 4.5).

1.4 ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY

In liquid column chromatography, the stationary phase is packed in a column. The sample,

which is usually a solubilized mixture of compounds including the analyte(s), is injected on the

top of the column and mobile phase flows continuously over the column. The sample

components will be distributed between the stationary and mobile phase. Because of a selective

delay on the stationary phase, the different sample components will elute with a different rate

through the column, resulting in sample separation. The component with the most affinity for

the stationary phase will be elute from the column as last. For the visualization of the

separation process a photodiode array (PDA) detector is used and a ultraviolet (UV)-

chromatogram is obtained.

Ultra performed liquid chromatography (UPLC) uses columns packed with small fully

porous particles (1.7 µm). The smaller particle size results in a higher back-pressure, requiring

specialized pump equipment. As a result of the smaller particle size, the Van Deemter curve is

flattened out, resulting in relative higher separation efficiencies with higher flow rates (Figure

1.3). The lower Height Equivalent of a Theoretical Plate (HETP), the better the separation

efficiency and the column performance is. The physical dimensions (i.e. length and diameter)

9

of a UPLC column are reduced in comparison with a high performance liquid chromatography

(HPLC) column, because of the increase in back-pressure and increased column performance.

This results in a relative smaller sample injection volume during UPLC analysis.

A photodiode array (PDA) detector is coupled to the UPLC equipment. The advantage of

this detector is that the absorbance within a variety of wavelengths are recorded, usually

ranging from 190 nm to 400 nm, resulting in a wavelength spectrum. This spectrum can then be

used for verification of sample identity as well as peak purity verification. As UPLC peak are

generally sharper than HPLC peaks and good chromatographic practices require a minimum of

15 data points per peak, the sampling rate of a PDA detector coupled to UPLC is set higher

(e.g. 20 Hz) then when coupled to HPLC equipment (e.g. 1 Hz) [24].

Figure 1.3: The Van Deemter curve for different particle sizes [25]

The advantages of UPLC over HPLC analysis are the shorter analysis time, the increased

resolution and higher sensitivity [24-26], but instead, nowadays, the system is overall more

costly and less robust.

10

2. OBJECTIVE

In the pharmacy, active pharmaceutical ingredients (API) sub-label claims in

pharmaceutically compounded capsules is already a hot discussion topic during the last few

years. However, no studies were conducted to investigate how much API loss there is, where

this API loss occurs, and what could be the reason or origin for this API loss. The objective of

this research is to answer these raised questions.

This thesis contains an experimental part, where three kinds of capsules will be

pharmaceutically compounded, i.e. hydrocortisone, triamcinolone and dexamethasone,

following the guidelines of TMF. The working area and equipments will be swabbed in order to

discover if an API loss occurs, and if so, to quantitate this loss. Swabbing areas will include

the desk, the capsulation device or gelulier, the measuring cylinder, the weighing device, the

mortar, the pestle, the flexible plastic card and the paper funnel. A UPLC-PDA method will be

used to determine the amount of API present in the capsule and at the swabbed areas and

equipment.

The second part of this thesis uses assay data of 17 different molecules, obtained from the

the internal quality system of the UZ Ghent and from a study examining sub-label claim API

content in capsule (Ringtest 4). These assay results include following variables: (i) molecule

(API), (ii) API concentration, (iii) number of capsules made, (iv) pharmacist and (v)

preparation material (e.g. mortar influence). The assay results of each molecule will be

modeled in function of the molecule structural descriptors, examining if a chemical molecule-

effect on the observed sub-label claim API contents in capsules exists.

11

3. MATERIALS AND METHODS

3.1 CHEMICALS

Methanol (MeOH, HPLC grade) and acetonitrile (ACN, UPLC quality) were obtained

from Fisher Chemical (Loughborough, United Kingdom). Water was purified using a Milli-Q

academic water purification system (Sartorius, Göttingen, Germany), resulting in <18.2

mΩ×cm quality. The API compounds hydrocortisone, triamcinolone and dexamethasone were

obtained from Fagron (Waregem, Belgium) and the excipient diluant B (99.5 % mannitol

powder and 0.5 % anhydrous colloidal siliciumdioxide) was obtained from Université de Liège

(Liège, Belgium). The colorant riboflavin was obtained from C.V. Laboratoria Flandria

(Zwijnaarde, Belgium). Sodium dihydrogen phosphate monohydrate (Merck acs, Darmstadt,

Germany) and di-potassium hydrogen phosphate anhydrous (Panreac Quimica sa, Barcelona,

Spain) were used to make the 10 mM phosphate buffer pH 4.

3.2 MATERIALS AND EQUIPMENTS

Following materials and equipment were used in this thesis:

- Sonification of solutions was done using Branson 2210 (A4 Ultrasone)

- UPLC analysis was done on a Waters Acquity H UPLC Class using a Quaternary

Solvent Manager, a Waters Acquity Sample Manager, combined with a Flow Through

Needle, and a Waters Acquity Performance LC PDA

- Centrifugation of the solutions was done using Eppendorf Centrifuge 5810R at 3220 x

g for 5 minutes at 22°C

- A Mettler Toledo balance (type:X5105DU) was used, with a sensitivity of 0.01 mg

- Finnpipette Thermo Scientific of 100-1000 µL

- Hard gelatin capsules (n°2, yellow) were obtained from Feton (Brussels, Belgium)

- Filter (Whatmann 1, 90 mm Ø, Cat No 1001090)

- HPLC glass vials

- Microscope (Axiostar plus, Zeiss) and a photo camera (Canon, power shot A640)

- Mortar 1 (medium used, section 3.4.3.1) was used in section 3.4.1, 3.4.2, 3.5 and 3.6.

12

3.3 METHODS

3.3.1 UPLC–method and verification

The general UPLC method used an Acquity UPLC BEH C18 (50 x 2.1 mm; 1.7 µm)

analytical column combined with a VanGuardTM

BEH C18 (1.7 µm) guard column. The

column and sample compartment temperatures were set at 30°C (± 3°C) and 10°C (± 3°C),

respectively. The flow rate was set at 0.5 mL/min and the injection volume at 2 µL. PDA

detection (190 - 400 nm) was used combined with a quantification wavelength of 240 nm. A

65/35 H2O/ACN mobile phase mixture was used for hydrocortisone and triamcinolone analysis,

whereas a 65/35 phosphate buffer pH 4 (10 mM)/ACN mobile phase was used for

dexamethasone analysis. A 50/50 H2O/MeOH sample solvent was used for hydrocortisone and

trimacinolone analysis, whereas a 70/30 H2O/MeOH dilution was used for dexamethasone

analysis.

For the method verification, linearity, LOQ and repeatability were investigated. For the

linearity, the concentration and peak area of reference 100%, 90% and 80% were used to

calculate the R2. For LOQ (mg/ml), reference 1% was used. The repeatability was evaluated

based upon independently prepared reference solutions.

3.4 HYDROCORTISONE CAPSULE PREPARATION

3.4.1 Hydrocortisone 10 mg capsules

3.4.1.1 Capsule preparation

Twenty capsules (n°2) containing 10 mg hydrocortisone were prepared according to TMF

[9].

3.4.1.2 Swabbing of working area and equipment + swab sample preparation

The capsulation device, the weighing device, the measuring cylinder, the paper funnel, the

desk and the mortar-pestle-flexible plastic card were swabbed using MeOH wetted cotton. The

swabs were transferred into a 50.0 mL volumetric flask and diluted with methanol to just below

volume. The flasks were sonicated for 5 min, diluted to volume using MeOH after which 10.0

mL was transferred into a 20.0 mL volumetric flask and diluted to just below volume using

H2O. The flasks were sonicated for 5 min and then diluted to volume using H2O. An aliquote

was transferred into a glass HPLC vial.

13

3.4.1.3 Preparation of reference solutions

Three different reference solutions (100%, 90% and 80% label claim) were made. Two 10

fold-dilution series resulting in 10%, 9% and 8% reference solutions and 1%, 0.9%, 0.8%

reference solutions were made. The final concentration of the 100% reference solution was

equal to 0.05 mg/mL hydrocortisone (50/50 MeOH/H2O).

3.4.1.4 Capsule sample preparation

Ten individual capsules, filled with the API powder mixture, were weighed. The capsules

were carefully opened and the powder content transferred into a 50.0 mL volumetric flask. The

emptied capsules were slightly closed, as some residual powder is still present, and were

weighed.

The powder mixture, obtained from the bulk capsule powder, was diluted to volume using

methanol and filtered. Of this filtrate, 25.0 ml was transferred into a 100.0 mL volumetric flask,

50.0 mL H2O was added and MeOH was used to dilute to just below volume. The flasks were

sonicated for 5 min and then diluted to volume using MeOH. An aliquote was transferred into a

glass HPLC vial.

3.4.1.5 Residual capsule sample preparation

The emptied capsules were re-opened and the two halves were carefully transferred into a

10.0 mL volumetric flask and dissolved in methanol. The flask was sonicated for 5 min to

dissolve the residual powder. 1.0 mL was transferred into a 2.0 mL volumetric flask and diluted

to just below volume using H2O. The flasks were sonicated for 5 min and then diluted to

volume using H2O. An aliquote was transferred into a glass HPLC vial.


The protocol given in section 3.4.1 is followed. The capsule sample preparation is

corrected for this increase in API content by additional dilution.

14

3.4.3 Mortar influence during hydrocortisone preparation

3.4.3.1 Different mortars

Four different mortars were evaluated. Details about these mortars are given in Table 3.1.

Table 3.1 Materials

Product description Supplier Condition

Mortar 1 HCT West Germany 181/125 Medium used

Mortar 2 Avignon France Porcelain, new, not used

Mortar 3 - Extensively used

Mortar 4 DruQuaR Agate for IR analyses

Pestle Omega pharma Same pestle was used in each preparation

Flexible plastic card - Same card was used in each preparation

3.4.3.2 Capsule preparation

In each mortar, two powder mixtures were prepared corresponding to the preparation of 20

capsules containing 10 and 20 mg hydrocortisone capsules (capsules n°2).

These eight powder mixtures were prepared according to the TMF guidelines. The last step

in which the powder mixture is uniformly distributed over the 20 capsules was not performed.

Here, the powder mixture present in the mortar will be transferred into a beaker instead.


The mortar, pestle and flexible plastic card were swabbed using MeOH wetted cotton. The

swabbing of this working equipments and the swab sample preparation are the same as in

section 3.4.1.2. The swabs sample preparations are diluted, so that their final concentrations are

located within the reference solution blocks.


The preparation of the hydrocortisone reference solutions is the same as in section 3.4.1.3.

15

3.5 TRIAMCINOLONE CAPSULE PREPARATION: 4 MG

The protocol similar to section 3.5 was followed. 20 capsules (n°2) of 4 mg triamcinolone

were made according to TMF [9]. The swabs sample preparations are diluted, so that their final

concentrations are located within the reference solution blocks.

3.6 DEXAMETHASONE CAPSULE PREPARATION: 0.25 MG

Some chromatography anomalies were seen, i.e. wide API peaks and presence of a

shoulder peak next to the main dexamethasone peak, when using the

hydrocortisone/triamcinolone UPLC method for dexamethasone assay. To improve the

chromatographic (UPLC) method, following changes were made: (1) the aqueous mobile phase

part was substituted for a 10 mM phosphate buffer pH 4, (2) the samples were diluted in 30/70

MeOH/H2O and (3) furthermore, it was decided so substitute the old guard column for a new

one. All these three “changes” are part of a good chromatography practices.

3.6.1 Influence of sample solvent and sample solvent strength

When using the hydrocortisone/triamcinolone UPLC method for dexamethasone assay, the

presence of a shoulder peak next to the main dexamethasone peak was noticed, see Figure 3.1.

Figure 3.1: Chromatography anomalies, i.e. wide API peaks and presence of a shoulder peak next

to the main dexamethasone peak (using the hydrocortisone/triamcinolone UPLC method for

dexamethasone assay)

16

To improve the chromatographic (UPLC) method, some changes were made, which results

in a better chromatography, see Figure 3.2.

Figure 3.2: Optimized method: chromatogram obtained using Acquity UPLC BEH C18 (50 × 2.1

mm; 1.7 µm) combined with new guard (lot: 0185302881) (0.003 mg/ml dexamethasone) (70/30

H2O/MeOH, mobile phase 35/65 ACN/10mM phosphate buffer pH 4)

The influence of different sample solvents, i.e. methanol and acetonitrile, on the overall

chromatography was investigated. Moreover, the sample solvent strength was also investigated.

For hydrocortisone and triamcinolone assay, a 50/50 MeOH/H2O sample solvent was used.

However, good chromatographic practices state that a weaker (or equivalent) sample solvent

strength, in comparison to (starting) mobile phase conditions, must be used [27].

Therefore, for dexamethasone assay, a 30/70 MeOH/H2O sample solvent was used. The

robustness of this sample solvent was checked by varying the ratios ± 10% (i.e. 20/80

MeOH/H2O and 40/60 MeOH/H2O).

3.6.2 Preparation of own-made 10%-dexamethasone trituration

3.6.2.1 Protocol 10%-dexamethsanone trituration

Due to the low quantity API needed (5 mg = 0.25 mg x 20), which is under the limit (50

mg), a trituration must be made. The own-made 10%-dexamethasone trituration was prepared

17

according to TMF of a comparable molecule, i.e. fludrocortisones acetate [9]. Following steps

were performed:

1: Weigh the amount of dexamethasone (100 mg), riboflavin (50 mg) and excipient

diluant B (100 mg) separately into a weighing device.

2: Calculate how much excipient diluant B is still required to obtain a 10 % trituration

and weigh this amount separately into a weighing device (1000 mg – x mg

dexamethasone – y mg riboflavin – z mg excipient diluant B).

3: Add 100 mg excipient diluant B in the mortar and crush/ground/mix with a pestle.

4: Add 50 mg riboflavin and mix until a homogeneous color is obtained.

5: Add 100 mg dexamethasone and mix again until a homogeneous color is obtained.

6: Add step by step (in small quantities) the rest of the excipient diluant B. Mix after

each addition to obtain an uniform colored mixture.

7: Weigh the container in which the trituration is to be placed.

8: Bring the content of the mortar in the container and weigh again, thus allowing the

calculation of the total trituration powder amount.


The weighing device, the measuring cylinder, the paper funnel, the desk+gloves, the

mortar, pestle and flexible plastic card were swabbed using MeOH wetted cotton. The swabs

were transferred into a volumetric flask. The swabs sample preparations are diluted and the

final concentration concentrations are located within the reference solution blocks.


Three different reference solutions (100%, 90% and 80%) were made. Then two 10 fold-

dilutions to 10%, 9% and 8% reference solutions and 1%, 0.9%, 0.8% reference solutions were

made. The 100% reference solution was equal to 0.003 mg/mL hydrocortisone (30/70

MeOH/H2O).

3.6.3 Analytical development and pilot recovery experiments

After initial 0.25 mg dexamethasone capsulations experiments (see section 3.6.4), an

unaccountable dexamethasone loss was noticed. Analytical development and pilot recovery

experiments were performed to locate the problem (see Fishbone-diagram in Attachment 1).

The objective was to eliminate the apparent, but yet unexplained, loss in dexamethasone at the

18

0.25 mg capsule preparation, i.e. to explain the inconsistent DEX mass balance. The influence

of the filtration and centrifugation (i.e. test one – three), of the mixing step (i.e. test a - d) and

of the swabbing technique (split-up versus combined) (i.e. test e and f) was evaluated. The

powder mixture (i.e. API and excipients) was representative for the preparation of 20 × 0.25 mg

dexamethasone capsules. An agate mortar and pestle were used.

3.6.3.1 Filter and centrifuge influence

The influence of filtration and centrifugation (5 min, 22°C, 3220 x g) in the sample

preparation on the dexamethasone assay was investigated.

A stock-concentration was made by transferring approximately 50.0 mg dexamethasone

into a 250.0 mL volumetric flask and diluted using methanol to just below volume. The flasks

were sonicated for 5 min, diluted to volume using MeOH.

Test one: Of the stock, 10.0 mL was transferred into a 100.0 mL volumetric flask and

diluted to volume using MeOH. 30.0 mL was transferred into a 100.0 mL volumetric

flask and diluted to just below volume using H2O. The flasks were sonicated for 5 min

and then diluted to volume using H2O. An aliquote was transferred into a glass HPLC

vial.

Test two: Of the stock, 10.0 mL was transferred into a 100.0 mL volumetric flask,

diluted to volume using MeOH and filtered/centrifuged. Of this filtrate/supernate, 30.0

mL was transferred into a 100.0 mL volumetric flask and diluted to just below volume

using H2O. The flasks were sonicated for 5 min and then diluted to volume using H2O.

An aliquote was transferred into a glass HPLC vial.

Test three: Similar to test b, but here an amount of excipient diluant B and riboflavin

was added in the 100.0 mL volumetric flask before filtration/centrifugation to imitate

the powder mixture present in the capsules.

3.6.3.2 Influence mixing step only

Test a: Transfer the trituration directly into a volumetric flask

Approximately 50 mg own-made 10%-dexamethasone trituration was transferred into

a 50.0 mL volumetric flask and dissolved/diluted to volume using MeOH. Then 2.0

mL was transferred into a 20.0 mL volumetric flask, diluted to volume using MeOH

and centrifuged (for 5 min at 22°C, 3220 x g). Of this supernate, 3.0 mL was

19

transferred into a 10.0 mL volumetric flask, diluted to volume using H2O. An aliquote

is transferred into a glass HPLC vial.

Test b: Trituration and excipient diluant B: from weighing device to volumetric flask

(in triplicate)

Approximately 50 mg own-made 10%-dexamethasone trituration was weighed into a

weighing device, which was transferred quantitative into a 50.0 mL volumetric flask.

At the same volumetric flask, the amount excipient diluant B needed for DEX capsule

preparation was added, to imitate the powder mixture present in the capsules. After

this, the weighing device was swabbed and the swabs were brought into the same

volumetric flask as the powder mixture. Following steps are similar to test a.

Test c: Trituration and excipient diluant B: from agaat mortar to volumetric flask

(powder mixture not chrushed/grounded/mixed with a pestle) (in triplicate)

Similar to test b, but here the trituration and excipient diluant B were first brought in

the agaat mortar (no crushing/grounding of the powder mixture) and then quantitative

transferred into a 50.0 mL volumetric flask. The mortar, card, desk and gloves were

swabbed as well and brought into the same volumetric flask as the powder mixture.

Test d: Trituration and excipient diluant B: from agaat mortar to volumetric flask

(powder mixture crushed/grounded/mixed with a pestle) (in triplicate)

Similar to test c, but here the powder mixture was crushed/grounded/ mixed with a

pestle. The pestle was swabbed as well and brought into the same flask as the powder

mixture.

3.6.3.3 Influence swabbing technique

Test e: Everything was brought together in one volumetric flask (different swabs +

powder)

The capsules were prepared following section 3.6.3.1 and the working equipments

were swabbed using MeOH wetted cotton. The different swabs and the powder

mixture was brought into a 50.0 mL volumetric flask. Following steps were similar

test a (section 3.6.3.2).

Test f: The swabs were splitted-up in different volumetric flasks

The capsules were prepared following section 3.6.3.1 and the working equipments

were swabbed using MeOH wetted cotton. The swabs were splitted-up in different

volumetric flasks and the swabs sample preparations are diluted, so that their final

20

concentrations are located within the reference solution blocks. The powder mixture

was transferred into a 50.0 mL volumetric flask and following steps are similar to test

a (section 3.6.3.2).

3.6.4 Dexamethasone 0.25 mg capsules

The capsules will be prepared, using the own-made 10%-dexamethasone trituration

(section 3.6.2) and using the commercial 10%-dexamethasone trituration (Fagron, Waregem,

Belgium).

3.6.4.1 Protocol capsule preparation

Twenty capsules (n°2) of 0.25 mg dexamethasone were prepared according to TMF of a

comparable molecule, i.e. fludrocortisones acetate [9]. Following steps were performed:

1: Weigh the amount of the dexamethasone trituration (5% overdosage is included to

compensate the loss during the preparation) and 1 g of excipient diluant B separately

into a weighing device.

2: Crush/ground/mix the excipient diluant B in the mortar with a pestle.

3: Add step by step the dexamethasone trituration and mix carefully after each

addition.

4: Bring the mixture in a measuring cylinder using a paper funnel, without ramming

the powder, and weigh the measuring cylinder.

5: Add, without ramming, excipient diluant B till calibration volume for capsules n°2

(addition of excipient diluant B q.s. ad 7.4 mL volume) and weigh the measuring

cylinder.

6: Bring the content of the measuring cylinder in the mortar and mix homogeneous.

7: Distribute the mixture uniform over the 20 capsules n°2.


The swabbing of the working equipments and the swab sample preparation are the same as

in section 3.6.2.2. Only here the capsulation device was swabbed as well.


The preparation of the dexamethasone reference solutions is the same as in section 3.6.2.3.

21

3.6.4.4 Capsule sample preparation

First ten individual capsules, filled with the API powder mixture, were weighed. Then the

capsules were very carefully opened and the powder content was transferred into a volumetric

flask. The emptied capsules were slightly closed, as some residual powder is still present and

were weighed. The samples are diluted in 30/70 MeOH/H2O and the final concentration was

approximately 0.003 mg/mL.

3.6.4.5 Residual capsule sample preparation

The emptied capsules were re-opened and the two halves were carefully transferred into a

volumetric flask. The samples are diluted in 30/70 MeOH/H2O and the final concentrations are

located within the reference solution blocks.

3.7 DATAMINING

The molecular structures of the 17 UZ Ghent and Ringtest 4 molecules were downloaded

from a chemical database (www.chemspider.com). These three-dimensional molecular

structures were optimized and used for calculating descriptors. A molecular descriptor is

defined as “the final result of a logic and mathematical procedure which transforms chemical

information encoded within a symbolic representation of a molecule into a useful number or

the result of some standardized experiment”. These descriptors can be used to interpret and

explain experimental data [28]. Structural descriptors are calculated through software and can

be classified into 5 blocks [29]:

- O D-descriptors: constitutional descriptors such as molecular weight and count

descriptors.

- 1 D-descriptors: functional group counts.

- 2 D-descriptors: topological descriptors.

- 3 D-descriptors: geometrical descriptor.

- Other: charge descriptors and molecular properties.

Constant descriptors, i.e. having no discriminating value, were eliminated. Each descriptor-

dataset was also corrected for molecular weight (dividing the individual uncorrected descriptor

values by the molecular weight of the corresponding compound). Finally, a z-score

normalization was performed:

22

In this equation, xi is the value of a data point, xm is the mean and SD is the standard

deviation of the data set. In this way, all descriptors are N (0,1) distributed.

Stepwise multiple linear regression (MLR) was used, each time a more specific descriptor

selection was made. Principal component analysis (PCA) was performed using all descriptors

(n=33) withheld in one or more MLR models. A partial least squares (PLS) model was

constructed, modeling the median assay values in function of the 33 withheld descriptors.

23

4. RESULTS AND DISCUSSION

4.1 METHOD VERIFICATION

The UPLC method used for HC, TRI & DEX assay was found to be linear (R2>0.99).

Repeatability was in compliance with in-house requirements (% RSD 2.67; n=9). Limit of

quantification, i.e. 2.55x10-5

mg/mL, indicated sufficient sensitivity for our API loss

investigation purposes.

4.2 HYDROCORTISONE


4.2.1.1 % label claim capsules

The % label claim was determined for ten randomly selected capsules and is given in

Figure 4.1, and was calculated as follows (Attachment 2):

Label claim API is expressed as a unit (amount API in one capsule), here 10 mg.

Figure 4.1: Percentage label claim of the ten individual HC 10 mg capsules

The average hydrocortisone assay, i.e. 92.16% LC (1.02% RSD), corresponds to previous

large scale hydrocortisone assay results obtained from different pharmacists: indeed, Ringtest 4

gave an average value of 92.87% LC (3.89% RSD).

80

85

90

95

100

1 2 3 4 5 6 7 8 9 10

% la

be

l cla

im

24

4.2.1.2 API mass balance

The total amount of hydrocortisone, recovered in the capsules and swabs is compared to

the hydrocortisone amount initially weighed before capsule preparation. For this, the total

hydrocortisone mass recovered in the 10 capsules was extrapolated to 20 capsule, as 20

capsules were prepared in total. Details regarding this API mass balance are given in Table 4.1.

The % API mass balance is calculated as follows:

.

Here the theoretical amount API is the total amount API initially weighted.

Table 4.1 API mass balance overview (10 mg hydrocortisone capsules)

Description Recovered HC mass (mg) API mass balance (%)1

20 Capsules

Bulk powder HC 181.72 90.01

Residual HC 2.59 1.28

Weighing device 0.85 0.42

Measuring cylinder 0.61 0.30

Mortar, pestle and flexible plastic card 14.10 6.98

Capsulation device 1.02 0.51

Desk 0.04 0.02

Overall 200.93 99.52

1: total amount hydrocortisone initially weighed, i.e. 201.9 mg

A total API mass balance of 99.52% was obtained. From this API mass balance, it can be

seen that the vast majority of the API loss occurs at the mortar, pestle and flexible card. In total,

84.86% of the observed API losses was seen at this step.



The % label claim was determined for ten randomly selected capsules (Attachment 3) and

is given in Figure 4.2.

25

Figure 4.2: Percentage label claim of the ten individual HC 20 mg capsules

The average hydrocortisone assay is 91.97% LC (2.20% RSD).


The total amount of hydrocortisone, recovered in the capsules and swabs are compared to

the hydrocortisone amount initially weighed before capsule preparation. Details regarding this

API mass balance are given in Table 4.2.

Table 4.2 API mass balance overview (20 mg hydrocortisone capsules)

Description Recovered HC mass (mg) API mass balance (%)1

20 Capsules

Bulk powder HC 363.19 90.48

Residual HC 4.69 1.17



Mortar, pestle and flexible plastic card 19.93 4.97


Desk 0.23 0.06

Paper funnel 0.04 0.01

Overall 391.11 97.44

1: total amount hydrocortisone initially weighed, i.e. 401.39 mg


seen that the vast majority of the API loss occurs at the mortar, pestle and flexible card, as was

80

85

90

95

100

1 2 3 4 5 6 7 8 9 10

% la

be

l cla

im

26

also the case with the 10 mg hydrocortisone capsules. In total, 85.80% of the observed API

losses was seen at this step.

4.2.2.3 Comparison of hydrocortisone concentration in bulk and residual powder

In pediatrics and geriatrics, capsules are often opened (i.e. children cannot swallow the

capsules), so the amount API that remains at the capsule surface plays an important role. As

can be seen from previous sections, the global API content inside a capsule was calculated from

(i) the bulk powder (section 3.4.1.4) and from (ii) the residual powder sticking to the inside of

the capsule (section 3.4.1.5). The hydrocortisone concentration in both the individual bulk and

residual powders was calculated in order to detect a specific absorption phenomenon

(Attachment 4). In order to dissolve the residual powder of the individual capsules, the capsules

are immersed in MeOH, rendering the weighing of the capsules after this extraction process

impossible. Therefore, the average weight of 30 empty capsules (i.e. did not came into contact

with powder mixture) was taken as the weight of an empty capsule and was found to be 57.863

mg (RSD:1.57%). The HC concentration in bulk powder and in residual powder (%) is given in

Figure 4.3.

Figure 4.3: HC concentration in bulk powder and residual powder (%)

The average hydrocortisone concentration, in bulk powder is characterized by a very

narrow 95% confidence interval, due to low variability in both hydrocortisone amount

recovered (RSD: 2.16%) as well as the weight of the bulk powder (RSD: 1.67%).

The average hydrocortisone concentration in residual powder has a broader 95%

confidence interval, due to higher variability in both hydrocortisone amount recovered (RSD:

18.38%) as well as the weight of the bulk powder (RSD: 18.81%).

0

2

4

6

8

10

12

HC

co

nce

ntr

atio

n in

(b

ulk

/re

sid

ual

) p

ow

de

r (%

)

HC conc in bulk powder (%) HC conc in residual powder (%) error bar: 95% confidence interval

27

A statistically significant higher hydrocortisone concentration in the bulk powder than in

the residual powder is observed. Thus, those appears to be no selective concentration of

hydrocortisone occurring at the capsule surface. On the contrary, an concentration of

hydrocortisone in the bulk powder is observed, and a concentration of diluents to the capsule

surfaces. The amount API that sticks at the capsule shell (i.e. approximately 1%) is important

for the pediatrics and geriatrics, so they can take that information into account. Our data

indicates that this “loss” is not a specific HC loss but rather a powder-loss.

4.2.3 Concentration effect

The quantification and % API mass balance of 10 mg hydrocortisone capsules versus 20

mg hydrocortisone capsules can be found in Table 4.1 en Table 4.2. Theoretically, the total

powder mixture volume of both preparations should be constant (i.e. 20×0.37 mL), therefore

the hydrocortisone concentration in the 20 mg capsules powder mixture should be two-fold

higher than in the 10 mg capsules powder mixture. The influence of this difference in API

concentration on the different API loss steps is investigated.

If there is no selective hydrocortisone loss/adsorption (20 vs. 10 mg capsules):

recovered hydrocortisone mass (mg) x 2

hydrocortisone mass balance (%) is identical/similar

If there is a selective hydrocortisone loss/adsorption (20 vs. 10 mg capsules):

recovered hydrocortisone mass (mg) differs from x 2

hydrocortisone mass balance (%) differs/is not similar

In order to determine whether a concentration-effect is present at the different loss steps, it

is better to consider the mass balance approach as this already compensates for the different

amounts of hydrocortisone initially weighed.

The amount of API recovered from the 10 and 20 mg capsules (i.e. from bulk +

residual powder), differs by approximately a factor 2 and hence, the % API mass

balance is very similar between both concentrations.

The absolute API mass loss observed at the capsulation device has approximately

doubled and thus the % API mass balance remains the same. Therefore, a

concentration-effect is present at this step: the API mass balance will be approximately

similar for both dosages, hence API loss for 20 mg dosage will be higher.

28

The weighing device does not show this concentration-effect. This can be rationalized

by the fact that only a single area of the weighing device comes into contact with the

API powder, independently of the amount API weighed.

The API loss at the mortar (pestle + flexible plastic card) does not have a direct

proportional relationship to the hydrocortisone dosage increase. The API loss observed

at this step with the 20 mg hydrocortisone powder mixture is only 70.67 % from the

theoretical loss value as extrapolated from the API loss seen with the 10 mg

hydrocortisone powder mixture

). A possible hypothesis for

this observation is that API loss at the mortar surface, which is most likely a physical

phenomenon, see section 4.2.4, can be satisfied by API powder.

The desk area swabbed during the 10 mg hydrocortisone capsules preparation (20×50

cm = 1000 cm²) was approximately 5.5 times smaller than the desk area swabbed in 20

mg hydrocortisone capsules (100×55 cm = 5500 cm²). When taking this difference in

surface area in to account, a similar absolute mass loss is observed on the desk (0.04

mg x 5.5 ~ 0.22 mg).

The paper funnel cannot be compared due to the lack of an paper funnel in the 10 mg

hydrocortisone capsules. However, the loss at this point was very minimal.

The measuring cylinder used in the 10 mg hydrocortisone capsules differs from the

measuring cylinder used in the 20 mg hydrocortisone capsules. So the results of the

measuring cylinder cannot be compared.

4.2.4 Mortar influence

From sections 4.2.1.2 and 4.2.2.2 can be seen that, the largest API loss occurs at the

mortar. Therefore, this mortar variable was investigated further, by comparing four mortars,

differing in mortar material and mortar usage (Attachment 5). The % API mass balances

obtained from the different swabs for each preparation are given in Figure 4.4 and 4.5. For

objectively interpreting these results, a statistic analysis was performed using a chi² goodness-

of-fit test, see Table 4.3.

29

Figure 4.4: Hydrocortisone API mass balance during 10 mg capsule preparation

Figure 4.5: Hydrocortisone API mass balance loss during 20 mg capsule preparation

Table 4.3 Statistical analysis

Description 10 mg HC 20 mg HC

χ2

p-value χ2

p-value

Pestle 0.08 0.995 0.99 0.803

Card 0.15 0.985 0.62 0.891

Mortar 6.08 0.108 20.9 0.00011

Overall 2.55 0.466 12.21 0.00669

Significance level is 0.05. P-values < 0.05 are in bold and underlined.

0

1

2

3

4

5

6

7

8

M4 M2 M1 M3

% H

C m

ass

bal

ance

Overall Card Pestle Mortar

0

1

2

3

4

5

6

7

8

M4 M2 M1 M3

% H

C m

ass

bal

ance

Overall Card Pestle Mortar

30

From these figures, it is clear that significant hydrocortisone API mass loss occurs at the

mortar step. The % API loss at the card and pestle is fairly constant. The variability in overall

% API mass balance is mainly dependent on the mortar.

The results demonstrate that mortar 4 (agate mortar) has the lowest overall API mass

balance and mortar 3 (old mortar) has the highest overall API mass balance. In mortar 2 (new

mortar) the % API loss from the three different swabs (i.e. card, pestle and mortar) are

approximately the same.

The API loss as a function of the mortar (i.e. surface roughness) is given in Figure 4.6.

Figure 4.6: Percentage API loss of the mortar as a function of surface roughness

Two conclusions are made from Figure 4.6. The first conclusion is that the type of material

has an influence on the API mass balance, here the agate mortar has a lower API mass balance

than the porcelain mortar, even a new one. This is probably due to physical differences, but

chemical interactions cannot be ruled out. The second conclusion is that the used state of the

mortar also plays an important role on API loss, here the old, extensively used mortar has

approximately a five times higher API loss than the new, not used mortar. As the type of mortar

material is the same, this difference can only be attributed to a physical phenomenon in which

the powder mixture fills the space of the small scratches on the mortar surface. These scratches

are caused by the friction between pestle and mortar during mixing, in which certain hard

crystalline powders can cause additional stress on the mortar surface.

4.2.5 Suggestion for improvement of hydrocortisone capsule preparations

A suggestion of a new working instruction, which could possible reduce the % loss API

during hydrocortisone capsule preparations is given below:

0

1

2

3

4

5

6

M4 M2 M1 M3

% A

PI l

oss

mo

rtar

10 mg HC 20 mg HC

31

1: Calculate the mass excipient diluant B needed for the calibration volume for 20

capsules n°2 with the help of the density, instead of using a measuring cylinder to

determine the amount excipient diluant B needed for the calibration volume for 20

capsules n°2.

2: Place a paper under the capsulation device and place a paper at the bottom surface

area of the capsulation device on which the bottom part of the capsules are resting, to

catch any lost powder. Distribute the remaining powder mixture from the papers over

the 20 capsules n°2.

4.3 TRIAMCINOLONE

4.3.1 Triamcinolone 4 mg capsules




Figure 4.7: Percentage label claim of the ten individual TRI 4 mg capsules

The average triamcinolone assay, i.e. 96.90% LC (RSD: 2.38%), closely corresponds to

previous large scale triamcinolone assay results obtained from Ringtest 4 (97.0% (RSD:

4.39%)). The average triamcinolone assay value here is higher than the one obtain with

hydrocortisone, although identical compounding materials, e.g. mortar, pestle, etc., were used.

This can possibly be attributed to a molecule specific effect and will be investigated further in

section 4.5.

80

85

90

95

100

105

1 2 3 4 5 6 7 8 9 10

% la

be

l cla

im

32


The total amount of triamcinolone, recovered from the capsules and swabs are compared to

the triamcinolone amount initially weighed before capsule preparation. Details regarding this


Table 4.4 API mass balance overview (triamcinolone 4 mg)

Description Recovered TRI mass (mg) API mass balance (%)1

20 Capsules

Bulk powder TRI 76.72 95.64

Residual TRI 0.80 1.00



Mortar 1.85 2.31

Pestle 0.79 0.98

Flexible plastic card 0.19 0.24


Desk 0.02 0.03


Overall 81.57 101.68

1: total amount triamcinolone initially weighed, i.e. 80.22 mg

A total API mass balance of 101.68% was obtained. From this API mass balance, it is

shown that the vast majority of the API loss occurs at the mortar step. In total, 45.84% of the

observed API losses was seen at this step. As the average capsule label claim is above 95%, the

preparation of triamcinolone capsules is deemed to be under control, and no further

optimization of the production process is required.

33

4.4 DEXAMETHASONE

4.4.1 Own-made 10%-dexamethasone trituration

4.4.1.1 Dexamethasone content in own-made trituration

The dexamethasone content of the own-made trituration is given in Table 4.5. The % m/m

of the own-made dexamethasone triuration is calculated as follows:

Table 4.5: Dexamethasone content in the own-made trituration

# 10%-DEX trituration weighed (mg) DEX content (mg) (experimental) % (m/m)

1 50.35 5.07 10.08

2 50.75 4.98 9.81

3 50.56 5.07 10.03

Average 9.97

Stdev 0.14

%RSD 1.42

The mean dexamethasone concentration of the own-made trituration (n=3) is calculated to

be 9.97% (m/m).

4.4.1.2 Swabs dexametasone content and % API mass balance

The dexamethasone content in the different swabs (section 3.6.2.2) are given in Table 4.6.

The dexamethasone present in the trituration (mg) is calculated as follows:

.

The powder mass balance (%) is calculated as follows:

.

34

Table 4.6 % API mass balance (10% self made dexamethasone trituration)

Swab # Description Recovered DEX content (mg) % API mass balance1

1 Weighing device 0.77 0.15

2 Mortar 10.84 2.17

3 Pestle 6.09 1.22

4 Flexible plastic card 1.93 0.39

5 Desk + gloves 1.67 0.33

6 Trituration 492.16 98.45

Total 513.47 102.72

1: the total amount dexamethasone initially weighed. i.e. 499.89 mg

From these results, it is clear that the largest dexamethasone API mass loss occurs at the

mortar. A total API mass balance of 102.72% was obtained.

A powder mass balance of the 98.70% was found for the own-made 10%-dexamethasone

trituration, indicating limited losses of the overall powder mixture during the different

preparation steps.

4.4.2 Analytical development and pilot recovery experiments

4.4.2.1 Sample solvent strength influence

The influence of the different sample solvents and strengths, were evaluated based upon

the ratio of the small shoulder peak and large dexamethasone API peak. However, no statistical

significant effect could be seen for the use of different sample solvents and strengths, hereby

demonstrating the robustness of the sample solvent in dexamethasone assay method.

4.4.2.2 Filter and centrifuge influence

To investigate any additional API losses, which could explain the previously observed low

dexamethasone mass balance, the filter and centrifuge influence was investigated. The

influence of filtering or centrifuging the dexamethasone sample preparation is given in Table

4.7.

35

Table 4.7 Filter and centrifuge influence

Reference 100 % Filtration [relative (%)] Centrifugation [relative (%)]

unfiltered/uncentrifugated 100 100

filtered/centrifugated 102.08 100.39

+ excipient diluant B filtered/centrifugated 106.66 101.94

It can be seen that there is an up concentrating when filtering the samples. The peak area

obtained from the filtered 100% reference + excipient diluant B has the highest relative

concentration. The presence of excipient diluant B will cause the filtration to take longer,

allowing for the methanol to evaporate, which leads to an up-concentrating of dexamethasone.

Alternatively, centrifugation does not show this effect, and will be used in following

studies.

4.4.2.3 Influence swabbing technique

The results of the swabbing technique are given in Figure 4.8. From test a, the % m/m of

the own-made dexamethasone triuration was calculated (section 4.4.1.1) and used in test e to f

(Attachment 7).

Figure 4.8: influence swabbing technique: % API mass balance of test e and f

The % API mass balances in both tests (e and f) are approximately the same. Therefore,

analyzing the swabs separately has no influence on the dexamethasone mass balance.

4.4.2.4 Microscopy

The different powders used in the own-made dexamethasone trituration, i.e.

dexamethasone, excipient diluant B, riboflavin and the own-made and commercial

90

95

100

105

110

AP

I mas

s b

alan

ce (

%)

test e: everything in one volumetric flask test f: splitting up in different swabs

36

dexamethasone trituration powder visually investigated, using a 40X magnification, in order to

visualize the view (particle size and form) of these powders (see Figure 4.9).

Figure 4.9: Microscopic view powders (left above: dexametasone, middle above: excipient diluant

B, right above: riboflavin, left under: own-made trituration powder, right under: commercial

trituration powder)

From this figure, it can be seen that dexamethasone powder, the self made trituration

powder and the commercial trituration powder consist of little round particles, which stick

together. Excipient diluant B are quite large crystals. Riboflavin has the form of thin needles.

These fine needles stick together. A visual difference between the appearance of the own-made

trituration powder and the commercial trituration powder can be noticed.

4.4.2.5 Process: influence mixing step

The results of the influence of the mixing step (trituration+diluents) only are given in

Figure 4.10. From test a, the % m/m of the own-made dexamethasone triuration was calculated

(section 4.4.1.1) and used in test b to d (Attachment 7).

37

Figure 4.10: influence mixing step only: % API mass balance of test b, c and d

From these results, it’s clear that no significant API loss occurs when mixing

dexamethasone and the excipients.

These experiments evaluated the presence of untraceable dexamethasone loss during

capsule preparation. No such untraceable API loss was observed during the different steps in

the capsule preparation process. So the 0.25 mg dexamethasone capsules were prepared.

4.4.3 Dexamethasone 0.25 mg capsules using 10% own-made trituration

4.4.3.1 % label claim capsules (own-made trituration)



Figure 4.11: Percentage label claim of the ten individual DEX 0.25 mg capsules (own-made

trituration)

100

101

102

103

104

105

106

AP

I mas

s b

alan

ce (

%)

test b: trit + exc dil B ( from weiging device to volumetric flask)

test c: trit + exc dil B (from agaat mortar to volumetric flask, no mixing with a pestle)

test d: trit + exc dil B (from agaat mortar to volumetric flask, mixing with a pestle)

error bar: 95% CI

80

85

90

95

100

105

1 2 3 4 5 6 7 8 9 10

% la

be

l cla

im

38

The average dexamethasone assay, i.e. 98.00% LC (RSD: 1.74%), does not correspond to

previous large scale dexamethasone assay results obtained from Ringtest 4 (88.93% (RSD:

8.47%)). Furthermore, the 98.00% LC result underlines the importance of the dexamethasone

over-dosing step, as stipulated by TMF guidelines (section 3.6.4.1), in order to obtain capsules

containing the required API amount.

In a parallel study within Ringtest 4, a commercially available dexamethasone trituration

(10% m/m) was used, resulting in significantly better and less variable dexamethasone assay

results (96.87% (RSD: 4.16%)). The 0.25 mg dexamethasone capsules will be prepared using a

commercial trituration (section 4.4.4), so the obtained results can be compared with the results

of Ringtest 4.

4.4.3.2 API mass balance (own-made trituration)

The total amount of dexamethasone, recovered in the capsules and swabs are compared to

the dexamethasone amount initially weighed before capsule preparation. Details regarding this


Table 4.8 API mass balance overview (own-made trituration)

Description Recovered DEX mass (mg) API mass balance (%)1

20 capsules

Bulk powder DEX 4.85 89.91

Residual DEX 0.0482 0.89



Mortar 0.2052 3.80

Pestle 0.0586 1.09



Desk + gloves 0.0019 0.03


Overall 5.2632 97.54

1: total amount dexamethasone initially weighed, i.e. 5.40 mg

A total API mass balance of 97.54% was obtained. From this API mass balance, it is

confirmed that the vast majority of the API loss occurs in or at the mortar. In total, 56.47% of

the observed API losses was seen at this step.

39

4.4.4 Dexamethasone 0.25 mg capsules using a 10% commercial trituration

4.4.4.1 % label claim capsules (commercial trituration)



Figure 4.12: Percentage label claim of the ten individual DEX 0.25 mg capsules (commercial

trituration)

The average dexamethasone assay, i.e. 99.67% LC (RSD: 1.94%), closely corresponds to

previous large scale dexamethasone assay results obtained from Ringtest 4 (96.87% (RSD:

4.16%)).

4.4.4.2 API mass balance (commercial trituration)

The total amount of dexamethasone, recovered in the capsules and swabs are compared to

the dexamethasone amount initially weighed before capsule preparation. Details regarding this


80

85

90

95

100

105

1 2 3 4 5 6 7 8 9 10

% la

be

l cla

im

40

Table 4.9. API mass balance overview (commercial trituration)

Description Recovered DEX mass (mg) API mass balance (%)1

20 capsules

Bulk powder DEX 4.92 91.12

Residual DEX 0.0646 1.20



Mortar 0.1616 2.99

Pestle 0.0464 0.86



Desk + gloves 0.0022 0.04


Overall 5.30 98.27

1: total amount dexamethasone initially weighed, i.e. 5.40 mg


seen that the vast majority of the API loss occurs in or at the mortar. In total, 50.28% of the

observed API losses was seen at this step.

4.4.5 Comparison of the own-made and commercial trituration

Results the own-made trituration (section 4.4.3) and the results of the commercial

trituration (section 4.4.4) were compared.

Using a commercial trituration gave reasonable capsule assay values (99.67% LC), which

corresponds with the results obtained with the own-made trituration (98.00% LC). The loss

distribution of the swabs are given in Figure 4.13.

41

Figure 4.13: Loss distribution (left: own-made trituration and right: commercial trituration)

From Figure 4.13, it is clear that the loss distribution of the swabs in both triturations

(own-made and commercial) are similar. So no difference was noticed between the own-made

and commercial trituration.

4.5 INFLUENCE OF CHEMICAL STRUCTURE (DATAMINING)

4.5.1 Data

The data distribution of the assay results obtained with 17 different compounds were

investigated. In the Ringtest 4 study, certain preparations contain an overdose of API, therefore

a correction is made to these assay results. In order to eliminate other influences on the API

loss (e.g. mortar, API concentration, etc.), the median of the assay results for each molecule

was taken. This representative value will be modeled in function of the molecule’s descriptors.

A global overview of the 17 median assay values is given in Figure 4.14. Median values below

95% are indicted in red.

0,03

3,80

1,09

0,13

0,37

0,32

0,98 0,01 0,04

2,99

0,86

0,33

0,22

0,25

1,24

0,03 Desk + gloves

Mortar

Pestle

Card

Capsulation device Measuring cilinder Weighing device Paper funnel

42

Figure 4.14. Overview of median assay values (UZ Ghent and Ringtest 4) (no. of preparations is

given between brackets)

4.5.2 Modeling

A quantitative structure properties model (QSPR) is constructed expressing the observed

API content (i.e. median recovery value) in function of the molecules descriptors. Due to the

automatic removal of constant descriptors, 1604 descriptors remained of the original 3224

descriptors. This way, the complete data set was reduced to 1616 descriptors.

4.5.3 Stepwise multiple linear regression

4.5.3.1 Model 1: All selected descriptors (n=1616)

Stepwise multiple linear regression (MLR) is performed using the z-score normalised

descriptor values and z-score normalised molecular weight corrected descriptors. Probability of

F to enter was set to 0.05, for removal was set to 0.10. This gave a “full exhaustive model”

(with R²=1.000). When applying more strict F to enter/remove probabilities (i.e. 0.025 and

0.05), a model containing the three variables is obtained, characterized by a R² value of 0.925:

90

91

92

93

94

95

96

97

98

99

100

101

102

43

The summary of this model output (enter/removal: 0.025 and 0.05) is given Attachment 9.

This smaller model includes the RDF060m descriptor which was removed during the

construction of the first model (obtained with standard 0.05/0.10 F probability settings).

However, RDF085m was a relatively important descriptor in the “full-model”, with a similar

sign of coefficient (negative) and the highest standardized coefficient value i.e. -1.025. The

magnitude of the RDF060m coefficient is different from RDF085m coefficient, i.e. -1.172 vs. -

2.239. The R6e+ and R3m+ descriptors were also included within the full exhaustive model,

with similar coefficient signs and magnitude as in the smaller model. Descriptor explanation:

Radial distribution Function (RDF): “These descriptors are based on the distance

distribution in the geometrical representation of a molecule and constitute a radial

distribution function code that shows certain characteristics. The RDF in this form is

independent of the number of atoms (i.e. molecule size), unique regarding the 3D

arrangement of the entire molecule and invariant against translation and rotation of the

entire molecule” [30].

- RDF060m: Radial Distribution Function - 6.0 / weighted by atomic masses

- RDF085m: Radial Distribution Function - 8.5 / weighted by atomic masses

R-GETAWAY Autocorrelation descriptors (R): “This descriptor is based on spatial

autocorrelation formulas which weight the molecule atoms in such a way as to account for

atomic mass, polarizability, van der Waals volume, and electronegativity together with 3D

information encoded by the elements of the molecular influence/distance”[30].

- R6e+: R maximal autocorrelation of lag 6 / weighted by atomic Sanderson electro

negativities

- R3m+: R maximal autocorrelation of lag 3 / weighted by atomic masses

4.5.3.2 Model 2: selected descriptor groups (n=355)

A number of descriptor groups were selected. The summary of the model output is given in

attachment 10. The obtained model variables still cannot be directly related to a structural

characteristic of the molecule. A more selective descriptor screening is performed next.

4.5.3.3 Model 3: selected descriptors (n=55)

A number of descriptors, focusing on van der Waals interactions, polarity, dipole

influences and electrostatic descriptors, were selected. Summary of the obtained model is given

in Attachment 11.

44

4.5.4 Principal component analysis (PCA)

PCA was performed using all descriptors (n=33) withheld in one or more MLR models.

Explanatory R² values of the three PC are: 48.13%, 13.45% and 9.62%, respectively, or a

cumulative value of 71.20%. A PLS model was constructed, modeling the median assay values

in function of the 33 withheld descriptors. Score and loading plots are given in Attachment 12.

Explanatory and predictive R² and Q² values are given in Attachment 13. Clearly, the PLS

model indicates a strong influence of RDF060m and RDF085m, meaning the presence of heavy

(mass) atoms at a spherical distance of 6.0 Å and 8.5 Å from the geometrical centre of each

molecule. In general, higher RDF060m and RDF085m values are linked to compounds with

lower median recovery values. On the other hand, a higher hydration energy, i.e. energy

released when dissolving the compound in water, has a positive effect on the median assay

values. The hydration energy, combined with the lattice energy, determines whether or not a

compound dissolves in water (dissolves if hydration energy > lattice energy).

4.5.5 Discussion of models

One way ANOVA analysis of the individual molecule assay sets reveal a statistically

significant difference between the individual mean assay values of the 17 different molecules

(Attachment 14). By using the median of the complete assay data set for each molecule, an

assumption was made that other variables, including mortar condition at moment of use, are

smoothed out and that this is the best centrality estimate for the different data sets. Under this

assumption, this observed difference between the molecules mean assay values can only be

attributed to intrinsic molecule properties (be it chemical or physical). Post-hoc Tukey-Kramer

analysis of the ANOVA results indicates which molecules mean assay values differ statistically

significantly. A summary of the results are given in Attachment 14.

The obtained stepwise MLR model explains approximately 92.5% of the observed API

recovery variability within the 17 molecule data set. The obtained PCA model has a

comparable R² value of 0.95 and is described by 3 principal components. Both models indicate

that it is possible to model the 17 median assay values in function of intrinsic chemical

properties using only a limited set of factors. An overall average recovery of 95% is deduced

from the intercept of the model. The inter-molecule assay variability’s were compared using

homogeneity of variance test, i.e. Levene’s test (variance calculated relative to the mean value)

and Brown-Forsythe test (variance calculated relative to the median value). Both tests found

that the assay variability between the different molecules assay sets differs statistically

45

significant (Levene value: 9.079; Brown-Forsythe value: 23.660). This observation further

validates our modeling choice of selecting the median assay value for each molecule, as this

choice will, to some extent, remedy this observed difference in inter-molecule variability.

Section 4.1.4 demonstrated the influence of mortar usage on API (hydrocortisone) loss

during the mortar step. The importance of mortar usage (physical) and intrinsic molecule

(chemical) influence on API loss is summarized in Table 4.10.

Table 4.10 Comparison of mortar and molecule influence on API loss

Variable Condition Average %API loss Range (%)2

Porcelain mortar

(hydrocortisone)

new 1.24 1.31%

3.27 averagely used 2.55

1.96% extensively used 4.51

Molecule1

Acetylsalicylic Acid 03

8.72

Simvastatin 8.72

1 Best and worst molecules 2 Observed %API loss range between best and worst conditions 3 Median acetylsalicylic acid value: 100.52%, therefore %API mass loss was set at 0%

As previously mentioned, by using the median of the complete assay data set for each

molecule, we assume that operational variables, including mortar condition at moment of use,

are smoothed out. Therefore, we assume that the median assay value for the molecules were

obtained with an averagely used mortar (2.55% API loss). Table 4.10 demonstrates the

influence on % API loss when using a new or extensively used mortar, i.e. -1.31% and +

1.96%, respectively. This mortar variability on the loss (standard deviation: 1.65%, n=3),

expressing a purely physical phenomenon, is thus part of the observed variability around the

individual median values obtained from the 17 different molecules. Looking at the variability

within the assay data set obtained per molecule, we expect that the variability should be

characterized by a standard deviation of at least 1.65%, i.e. if only mortar variability is

considered. The variability within the assay sets of the individual molecules is depicted in

Figure 4.15.

46

Figure 4.15 Variability (as standard deviation) within individual molecule assay data (no. of

preparations is given between brackets – dashed line: mortar variability)

From this, it is clear that the expected mortar variability is indeed present within each of

the individual assay data sets of the 17 molecules. However, statistical analysis of the mortar

variance and the individual variances of the 17 molecules, only indicate a statistically

significant difference for fludrocortisone acetate (Levene’s test). This additional variability can

be attributed to other operational factors, and gives an indication of the molecules robustness

towards these operational factors. The larger the variability, the more sensitive the compound is

for these operational variables. Some examples of operational variables are no. of preparations

(n), mortar condition, API concentration, no. of capsules produced, personnel, capsulation

device.

4.6 FUTURE PERSPECTIVES

Estimation of the mortar condition (scratches, surface roughness,...). So that this

critical parameter can be kept under control. This should be preferably a single and

semi-qualitative limit test.

It would be interesting to investigate other variables as well, for example the

capsulation device (plastic and metal). In order to see if there is an influence on the

API loss. It might be that lower quality equipment would give lower recoveries.

0

1

2

3

4

5

6

7

8

9

10 St

and

ard

de

viat

ion

(%

)

47

5. CONCLUSION

The experimental part of this study confirms that there is an API loss present during the

capsule preparation. Of the three capsule preparations, hydrocortisone gave a loss of API assay

in the final capsules. The % label claim of hydrocortisone (±92%) was lower compared to the

% label claim of triamcinolone (±97%) and dexamethasone (±98%). For each of the three

molecules (hydrocortisone, triamcinolone and dexamethasone), the highest API loss was seen

at the mortar step (approximately 6.98% loss (HC), 2.31% loss (TRI) and 3.80% loss (DEX)).

Due to this observation, the mortar was investigated separately, using hydrocortisone as API.

From this study, a important role of the type of material and the used state of the mortar on API

loss was noticed. Two conclusions could be drawn from this experiment. First, use of the agate

mortar results in a lower API loss when compared to using a porcelain mortar. Secondly, the

older/more used the mortar becomes, the higher the API loss will be. Another interesting result

was that there was no concentration of the amount API at the capsule shell (approximately 1%),

which is important for the pediatric field. No difference between the own-made and

commercial dexamethasone trituration was noticed.

The influence of intrinsic molecular properties on the API loss observed during capsule

preparations was investigated using a dataset of 17 molecules, obtained from UZ Ghent and

from a previous ringtest study. Median assay values for the 17 molecules were calculated,

effectively smoothing out other variables during the capsule production process. ANOVA

analysis demonstrated the presence of a molecule (chemical) effect on the median assay values.

This molecule effect was modeled, by expressing the 17 median assay values in function of

corresponding molecular descriptors, using stepwise MLR and PCA techniques. The obtained

MLR model, containing 3 variables (RDF060m, R6e+ and R3m

+) explained 92.5% of the

observed % API loss variability, whereas the PCA model explained 95%.

Future research efforts are needed to improve the API loss during capsule preparation, i.e.

to decrease the loss as well as its variability.

48

6. REFERENCES

1. Art. 1 van de Belgische Wet van 25 maart 1964 op de geneesmiddelen.

2. Buren, C.v., Een korte geschiedenis van tweehonderd jaar geneesmiddelen en

apothekers. 2007.

3. Mignani, S., et al., Expand classical drug administration ways by emerging routes using

dendrimer drug delivery systems: A concise overview. Advanced Drug Delivery

Reviews, 2013.

4. Mrsny, R.J., Oral drug delivery research in Europe. Journal of Controlled Release,

2012. 161(2): p. 247-253.

5. Isha, C., et al., Oral sustained release drug delivery system: an overview. International

Research Journal of Pharmacy, 2012. 3(5): p. p. 57-62.

6. Merck Manual Medisch Handboek. 2 ed. 2005. 1970.

7. Elling, H., Inleiding in de farmacotherapie. 2009: Bohn Stafleu van Loghum.

8. European Pharmacopoeia 7th edition.

9. Therapeutisch Magistraal Formularium. uitgave 2010 ed.

10. Pharmaceutical Capsules. second ed. 288.

11. Al-Tabakha, M.M., HPMC Capsules: Current Status and Future Prospects. Journal of

Pharmacy and Pharmaceutical Sciences, 2010. 13(3): p. 428-442.

12. Cole, E.T., D. Cad, and H. Benameur, Challenges and opportunities in the

encapsulation of liquid and semi-solid formulations into capsules for oral

administration. Advanced Drug Delivery Reviews, 2008. 60(6): p. 747-756.

13. The Chicago Tylenol Murders. Available from: http://historicmysteries.com/the-

chicago-tylenol-murders/ (10-4-2013)

14. Magistrale bereidingen, in Test Gezondheid. December 2010/ Januari 2011. p. 18-22.

15. Arseus Haalt dit jaar 80 % omzet uit eigen merken, in De Gids voor de Beste Belegger.

Maart 2013. p. 26-31.

16. Capehart, K.D., A Laboratory Exercise in Capsule Making. American Journal of

Pharmaceutical Education, 2008. 72(5).

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Farmaco, 1999. 54(1-2): p. 1-14.

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blenders. Drug Dev Ind Pharm, 2002. 28(8): p. 905-18.

http://historicmysteries.com/the-chicago-tylenol-murders/

http://historicmysteries.com/the-chicago-tylenol-murders/

49

19. Bauman, I., D. Curic, and M. Boban, Mixing of solids in different mixing devices.

Sadhana-Academy Proceedings in Engineering Sciences, 2008. 33(6): p. 721-731.

20. Marshall, S.J., et al., A review of adhesion science. Dent Mater. 26(2): p. e11-6.

21. Susana, L., P. Canu, and A.C. Santomaso, Development and characterization of a new

thief sampling device for cohesive powders. International Journal of Pharmaceutics,

2011. 416(1): p. 260-267.

22. van der Velden, V.H., Glucocorticoids: mechanisms of action and anti-inflammatory

potential in asthma. Mediators Inflamm, 1998. 7(4): p. 229-37.

23. Cirillo, N., et al., Characterization of a novel oral glucocorticoid system and its

possible role in disease. J Dent Res. 91(1): p. 97-103.

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Method for Tolterodine Tartrate: Application in Pharmaceuticals, Human Plasma and

Urine Samples. Sci Pharm. 80(1): p. 101-14.

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pharmaceutical analysis. Talanta, 2006. 68(3): p. 908-918.

26. Wren, S.A.C., Peak capacity in gradient ultra performance liquid chromatography

(UPLC). Journal of Pharmaceutical and Biomedical Analysis, 2005. 38(2): p. 337-343.

27. Boonen, J., et al., A critical quality parameter in quantitative fused-core

chromatography: The injection volume. Journal of Pharmaceutical Analysis, (0).

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http://www.moleculardescriptors.eu/tutorials/what_is.htm (23-4-2013).

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Knowledge based on:

- Course Artsenijbereidkunde (prof C. Vervaet)

- Course Instrumentele Analytische Chemie (prof L. Thienpont)

http://www.moleculardescriptors.eu/tutorials/what_is.htm

7. ATTACHMENT

ATTACHMENT 1: FISHBONE-DIAGRAM

Figure 7.1 Fishbone-diagram

ATTACHMENT 2: HYDROCORTISONE 10 MG CAPSULES

Table 7.1: 10 mg hydrocortisone capsules: hydrocortisone content in capsules

Capsule # HC capsule

powder (mg)

Residual HC (mg)

in “empty”

capsules

Total

recovered HC

(mg)

% LC % API mass

balance

1 9.28 0.10 9.38 93.81 92.93

2 8.99 0.20 9.18 91.84 90.97

3 9.02 0.10 9.12 91.24 90.38

4 9.10 0.12 9.22 92.20 91.33

5 9.11 0.14 9.25 92.50 91.63

6 9.16 0.13 9.28 92.81 91.93

7 8.94 0.13 9.07 90.71 89.86

8 9.04 0.11 9.15 91.54 90.68

9 9.05 0.13 9.18 91.76 90.89

10 9.19 0.13 9.32 93.17 92.29

Average 9.09 0.13 9.22 92.16 91.29

%RSD 1.11 20.77 1.02 1.02 1.02

ATTACHMENT 3: HYDROCORTISONE 20 MG CAPSULES

Table 7.2: 20 mg hydrocortisone capsules: hydrocortisone content in capsules

Capsule # HC capsule

powder (mg)

Residual HC (mg)

in “empty”

capsules

Total

recovered HC

(mg)

% LC % API mass

balance

1 18.66 0.27 18.93 94.67 94.34

2 18.44 0.32 18.75 93.77 93.45

3 18.02 0.25 18.28 91.39 91.07

4 17.78 0.23 18.00 90.02 89.71

5 18.21 0.18 18.39 91.96 91.64

6 18.42 0.17 18.59 92.96 92.64

7 18.35 0.23 18.58 92.88 92.56

8 18.12 0.21 18.33 91.66 91.35

9 17.29 0.23 17.51 87.57 87.26

10 18.30 0.26 18.56 92.81 92.49

Average 18.16 0.23 18.39 91.97 91.65

%RSD 2.16 18.38 2.20 2.20 2.20

ATTACHMENT 4: HYDROCORTISONE CONCENTRATION IN BULK AND

RESIDUAL POWDER

Table 7.3: 20 mg hydrocortisone capsules: hydrocortisone concentration in bulk powder

Capsule # HC bulk powder (mg) Bulk powder (mg)1 HC conc in bulk powder (%)

2

1 18.66 163.36 11.42

2 18.44 168.16 10.96

3 18.02 164.75 10.94

4 17.78 161.72 10.99

5 18.21 169.74 10.73

6 18.42 167.36 11.01

7 18.35 166.42 11.02

8 18.12 169.23 10.71

9 17.29 162.63 10.63

10 18.30 166.39 11.00

Average 18.16 165.98 10.94

%RSD 2.16 1.67 2.04

95% CI of average HC conc. in bulk (%) 10.78 to 11.10

1: capsule weight (full) – capsule weight (without bulk powder)

2:

Table 7.4: 20 mg hydrocortisone capsules: hydrocortisone concentration in residual powder

Capsule # HC residual powder (mg) Residual powder (mg)1

HC conc in residual powder

(%)2

1 0.27 4.00 6.78

2 0.32 3.44 9.21

3 0.25 4.03 6.30

4 0.23 1.95 11.67

5 0.18 3.05 5.92

6 0.17 3.18 5.40

7 0.23 4.21 5.46

8 0.21 3.49 6.00

9 0.23 3.80 5.95

10 0.26 3.55 7.27

Average 0.23 3.47 7.00

%RSD 18.38 18.81 28.40

95% CI of average HC conc. in residual (%) 5.58 to 8.42

1: capsule weight (without bulk powder) – capsule weight (empty)

Capsule weight empty: 57.863 mg (%RSD: 1.57%)

2:

ATTACHMENT 5: MORTAR INFLUENCE

Table 7.5: Mortar influence: hydrocortisone content in swabs in mortar 1: DruQuaR

Swab # Description HC content (mg)

(10 mg HC caps.)

%API

mass

balance

HC content (mg)

(20 mg HC caps.)

% API

mass

balance

1 Flexible plastic card 2.80 1.39 3.12 0.78

2 Pestle 2.68 1.33 4.30 1.07

3 Mortar 4.95 2.46 10.59 2.46

Overall 10.43 5.18 18.00 4.49

Table 7.6: Mortar influence: hydrocortisone content in swabs in mortar 2: New


(10 mg HC caps.)

% API

mass

balance

HC content (mg)

(20 mg HC caps.)

% API

mass

balance


2 Pestle 2.84 1.42 3.94 0.98

3 Mortar 2.53 1.26 4.89 1.22

Overall 7.49 3.73 13.23 3.30

Table 7.7: Mortar influence: hydrocortisone content in swabs in mortar 3: Old


(10 mg HC caps.)

% API

mass

balance

HC content (mg)

(20 mg HC caps.)

% API

mass

balance


2 Pestle 2.29 1.14 6.59 1.65

3 Mortar 7.72 3.84 20.69 5.17

Overall 12.06 6.00 30.18 7.54

Table 7.8: Mortar influence: hydrocortisone content in swabs in mortar 4: Agate


(10 mg HC caps.)

% API

mass

balance

HC content (mg)

(20 mg HC caps.)

% API

mass

balance


2 Pestle 2.39 1.19 4.08 1.02

3 Mortar 1.17 0.59 2.22 0.55

Overall 5.96 2.98 10.94 2.73

ATTACHMENT 6: TRIAMCINOLONE 4 MG CAPSULES

Table 7.9 Triamcinolone content in capsules

Capsule # TRI capsule

powder (mg)

Residual TRI (mg)

in “empty”

capsule

Total TRI

(mg) % LC

% API mass

balance

1 3.81 0.04 3.84 96.12 95.86

2 3.92 0.06 3.99 99.68 99.41

3 3.97 0.04 4.01 100.17 99.90

4 3.90 0.05 3.95 98.69 98.42

5 3.81 0.04 3.85 96.17 95.91

6 3.73 0.05 3.77 94.29 94.03

7 3.91 0.04 3.95 98.68 98.41

8 3.77 0.03 3.79 94.77 94.51

9 3.71 0.03 3.75 93.68 93.42

10 3.84 0.03 3.87 96.77 96.50

Average 3.84 0.04 3.88 96.90 96.64

%RSD 2.26 27.24 2.38 2.38 2.38

ATTACHMENT 7: DEXAMETHASONE: SIX EXPERIMENTS

Table 7.10: Test b: API mass balance (%)

#

10%-DEX

trituration weighed

(mg)

DEX content (mg)

present in the

trituration

(theoretical)1

DEX content

recovered (mg)

(experimental)

% API mass

balance2

1a 50.77 5.06 5.17 102.07

2b 50.14 4.81 5.00 103.98

3b 50.06 4.80 4.95 103.05

Average 103.03

Stdev 0.955

% RSD 0.927

a: DEX trituration concentration: 9.97% m/m (analyzed on 25/4/2013)

b: DEX trituration concentration: 9.59% m/m (analyzed on 26/4/2013)

Table 7.11: Test c: API mass balance (%)

#

10%-DEX

trituration weighed

(mg)

DEX content

present in the

trituration (mg)

(theoretical)1

DEX content

recovered (mg)

(experimental)

% API mass

balance2

1a 50.68 5.05 5.25 103.81

2b 49.95 4.79 4.98 103.85

3b 50.15 4.81 5.03 104.55

Average 104.07

Stdev 0.416

% RSD 0.400



Table 7.12:Test d: API mass balance (%)

#

10%-DEX

trituration weighed

(mg)

DEX content

present in the

trituration (mg)

(theoretical)1

DEX content

recovered (mg)

(experimental)

% API mass

balance2

1a 50.72 5.06 5.34 105.54

2b 49.90 4.79 4.94 103.24

3b 49.85 4.78 4.96 103.64

Average 104.14

Stdev 1.229

% RSD 1.180



Table 7.13: Test e and f: API mass balance (%)

#

10%-DEX

trituration weighed

(mg)

DEX content

present in the

trituration (mg)

(theoretical)1

DEX content

recovered (mg)

(experimental)

% API mass

balance2

Test ea

54.64 5.20 5.53 106.22

Test fb

54.66 4.23 4.52 107.01

a: DEX trituration concentration: 9.52% m/m (analyzed 2/5/2013)

b: DEX trituration concentration: 7.73% m/m (analyzed 3/5/2013)

ATTACHMENT 8: DEXAMETHASONE 0.25 MG CAPSULES

Table 7.14: Dexamethasone content in capsules (own-made trituration)

Capsule # DEX bulk powder

(mg)

Residual DEX

(mg)

Total DEX

(mg) %LC

% API mass

balance

1 0.240 0.003 0.242 96.94 89.83

2 0.241 0.002 0.243 97.18 90.05

3 0.244 0.002 0.246 98.56 91.33

4 0.241 0.002 0.244 97.43 90.28

5 0.241 0.002 0.244 97.49 90.34

6 0.239 0.003 0.242 96.71 89.62

7 0.246 0.002 0.248 99.12 91.85

8 0.238 0.003 0.241 96.33 89.27

9 0.242 0.003 0.245 97.97 90.78

10 0.253 0.002 0.256 102.23 94.73

Average 0.243 0.002 0.245 98.00 90.81

%RSD 1.80 8.24 1.74 1.74 1.74

; LC: label claim DEX, i.e. 0.25 mg

% API mass balance:

; theoretical DEX in one capsule (mg):

Table 7.15: Dexamethasone content in capsules (commercial trituration)

Capsule # DEX bulk powder

(mg)

Residual DEX

(mg)

Total DEX

(mg) %LC

% API mass

balance

1 0.238 0.003 0.242 96.73 89.59

2 0.246 0.004 0.250 99.95 92.58

3 0.242 0.002 0.244 97.61 90.41

4 0.250 0.003 0.253 101.32 93.85

5 0.248 0.003 0.251 100.42 93.01

6 0.250 0.004 0.254 101.49 94.01

7 0.244 0.002 0.246 98.46 91.19

8 0.239 0.005 0.244 97.58 90.38

9 0.249 0.003 0.252 100.87 93.43

10 0.252 0.003 0.256 102.26 94.71

Average 0.246 0.003 0.249 99.67 92.32

%RSD 1.99 22.74 1.94 1.94 1.94

; LC: label claim DEX, i.e. 0.25 mg

% API mass balance:

; theoretical DEX in one capsule (mg):

ATTACHMENT 9: DATAMING: SUMMARY MODEL 1 OUTPUT

Table 7.16. Stepwise MLR using z-score normalised descriptors. (enter/removal: 0.025 and 0.05)

Variable

Unstand. coefficients Standardized coeff.

R² F value

Coefficient St. error Beta

Constant 95.409 0.161 -

0.925 53.768

RDF060m -1.172 0.215 -0.536

R6e+ 1.278 0.240 0.585

R3m+ -0.701 0.204 -0.321

ATTACHMENT 10: SUMMARY MODEL 2 OUTPUT

Probability of F to enter was set to 0.05, for removal was set to 0.10.

Table 7.17. Stepwise MLR using z-score normalised descriptors.

Variable

Unstand. coefficients St. coeff.

R² F value

Coefficient Standard error Beta

Constant

G2

95.409

-1.693

0.346

0.357

-

-0.775 0.600 22.514

Table 7.18. Stepwise MLR using z-score normalised molecular weight corrected descriptors.

Variable


R² F value


Constant

PW5

Neoplastic-80

HyDp

95.409

2.446

1.158

0.729

0.194

0.291

0.220

0.292

-

1.119

0.530

0.334

0.891 35.434

Descriptor explanation:

G2: gravitational index G2 (bond-restricted)

PW5: path/walk 5 - Randic shape index

Neoplastic-80: Ghose-Viswanadhan-Wendoloski antineoplastic-like index at 80%

HyDp: hyper-distance-path index

ATTACHMENT 11: SUMMARY MODEL 3 OUTPUT

Probability of F to enter was set to 0.05, for removal was set to 0.10.

Table 7.19. Stepwise MLR using z-score normalised descriptors (standard F probability)

Variable


R² F value


Constant

G2

95.409

-1.693

0.346

0.357

-

-0.775 0.600 22.514

As only 1 variable is selected in the model (Table 7.15), the standard F probability values are

altered to allow more variables to enter the model. Probability of F to enter was set to 0.15,

for removal was set to 0.30. Summary of the obtained model is given in Table 7.16.

Table 7.20. Stepwise MLR using z-score normalised descriptors (adjusted F probability)

Variable


R² F value


Constant

G2

nHBonds

ALogP2

nHDon

Hydratation Energy

MLogP

95.409

-0.339

-0.587

-2.069

-2.789

-1.907

0.628

0.175

0.288

0.194

0.376

0.603

0.486

0.274

-

-0.155

-0.269

-0.947

-1.276

-0.873

0.287

0.932 22.736

Table 7.21. Stepwise MLR using z-score normalised molecular weight corrected descriptors.

(standard F probability)

Variable


R² F value


Constant

Ms

nHBonds

AROM

S

95.409

2.491

-0.520

1.174

-1.668

0.235

0.556

0.255

0.325

0.627

-

1.140

-0.238

0.537

-0.763

0.852 17.304

Descriptor explanation:

G2 or gravitational index G2 (bond-restricted) ranges from 7 to 21 in the original

descriptor matrix. This descriptor represents the uniformity of mass distribution

throughout the molecule. However, from the calculation it can be seen that this G2

descriptor is correlated with the molecular weight of the molecules (R² of 0.958 when

plotting MW vs. G2). Therefore, the entire descriptor set was corrected for the molecular

weight.

Ms or mean electrotopological state (Si)

nHBonds or number of hydrogen bonds ranges from 0 to 1 and is based on the possible

number of intramolecular hydrogen bonds.

AROM or aromaticity index ranges from 0 to 0.93 in the original descriptor matrix. It is

calculated based on the bond lengths and bond orders of the compounds.

S or the Abrahams dipolarity/polarizability descriptor ranges from 0.25 to 2.86 in the

original descriptor matrix.

ATTACHMENT 12: SCORE AND LOADING PLOTS PCA AND PLS

Figure 7.2. Score plot of PCA analysis

Figure 7.3. Loading plot of PCA analysis.

Figure 7.4. Score plot of PLS model

Figure 7.5. Loading plot of PLS model

ATTACHMENT 13: EXPLANATORY (R²) AND PREDICTIVE (Q²) PCA MODEL

VALUES

Figure 7.6 Explanatory (R²) and predictive (Q²) PCA model values

ATTACHMENT 14: ANOVA

Table 7.22 One way ANOVA using assay data sets from the 17 different molecules

Sum of Squares df Mean Square F Sig.

Between groups 4621.524 16 288.845 23.515 0.000

Within groups 23399.517 1905 12.283 - -

Total 28021.041 1921 - - -

Note: The 17 mean assay values are within the same range as the corresponding 17 median assay values (i.e. ± 1%)

Table 7.23 Post hoc ANOVA analysis by Tukey-Kramer method

A

SA

All

op

.

Bu

tylh

.

Da

ps.

Dexa

.

Flu

dro

.

Fo

lic

A.

Fu

ro.

Hy

dra

l.

Hy

dro

ch

l.

HC

Ph

eno

b.

Pred

n.

Pro

pr.

Sim

v.

Sp

iro

no

Tria

m.

ASA X X X X X X X X X X X X X X X

Allop. X X X X X X X X

Butylh. X X

Daps. X X X

Dexa. X X X X X X X X X

Fludro. X X X X X X

Folic A. X X X X X X X X

Furo. X X X X X X X X X X X

Hydral. X X X X X X X X

Hydrochl. X X X X X X X X

HC X X X X X X X X

Phenob. X X X X X X X X X

Predn. X X X X X

Propr. X X X X

Simv. X X X X X X X X X X X X X X

Spirono. X X X X X X

Triam. X X X X

X: statistically significant difference (0.05 significance level)

8. EVENING LECTURES: INTERNATIONALISATION AT HOME

Evening lecture 1: “Biedt het kiwi-model een oplossing voor de financiële problemen van

de sociale zekerheid?” by Dirk Duppen

The Kiwi-model was originated in New Zealand. Here the government will look at what

is needed, they will perform a scientific need-analysis and based on evidence-based medicines

(EBM), they choose the best product. After which, they will use the combined purchasing

power of the community to agree a correct price with the companies through a public

quotation request. Since Dirk Duppen has released his book “de cholesteroloorlog”, in which

he pleads for the Kiwi-model, more attention is paid to the Kiwi-model in Belgium. Thanks to

the Kiwi-model: for the same drugs, some huge price differences were noticed.

My opinion about the Kiwi-model is that it is well thought and that interesting results can

be obtained. It’s good that the cost of the medicines can be brought down and that the

obstacles in the medicine policy can be handled. For example the fall in price of the human

papillomavirus-vaccine provides a higher vaccination degree, which is good for the

healthcare. So I’m for a Kiwi-model in Belgium. I was also surprised that a lot of studies hold

back information or manipulate their study to get a good result. I didn’t thought that this

happened in so many studies.

Evening lecture 2: “Registration of new vaccines in the EU: not an easy task.” by Pieter

Neels

This evening lecture was about the importance of vaccines and that they are different

from medicines. They are extremely efficient and have a high impact on the health. A vaccine

is mostly given to healthy people (often to young infants) and they have a preventive action,

they are not used to cure a disease. Vaccines must have a good quality and be safe to meet the

requirements. An ideal vaccine has a high efficiency, is safe, has no side effects (which is

almost impossible), has a long protection time, exist preferably out of one dose, is preferably

at room temperature and is as cheap as possible. There exist different types of vaccines,

namely live vaccines and non living vaccines. And still new vaccines are in progress.

I’m for vaccination, because I think it reduces the mortality and that some severe diseases

could disappear. It’s important to vaccinate your child, because it protect your child against

diseases like measles, mumps, rubella, tetanus, hepatitis, … I find it less terrible to have some

side-effects of the vaccine, then to undergo the disease.

Evening lecture 3: “Innovating for a better and sustainable heathcare” by Rudi Pauwels

Rudi Pauwels is a pharmaceutical scientist. He founded already a few companies: namely

Tibotec-Vicro, Galapagos and Biocartis. On March 22, 2013 he obtained a honoray doctorate.

He is a HIV researcher and

Rudi Pauwels talked about how each people is different and that we need to become more

personal and detach from the idea that one drug will be good for a lot of diseases. There is a

large genetically diversity. It is important to know why people are sick and what are the

causes of mortality. He also talked about his latest product Apollo, which is a diagnostic

platform.

This was an interesting lecture. I agree with Rudi Pauwels that way of treatment needs to

be changed, it needs to become more patient-related.

Evening lecture 4: “Hospital admissions related to medication” by Patricia van den

Bremt

In this lecture Patricia van den Bremt talked about how drugs can harm us. There are two

ways in which a drug can harm: intrinsic and extrinsic toxicity. The intrinsic toxicity means

that there are always side effects (ADR=Adverse Drug Reaction) which are non-avoidable

and which are no errors. With extrinsic toxicity, they means the errors were you do something

wrong with the drug (wrong dosage, wrong indication). Such medication errors can harm us

(not necessary) and such errors are avoidable. An adverse drug event (ADE) is a combination

of ADR (non-avoidable harm) and medication errors (avoidable harm). It is important to have

clear definitions so no confusion can occur and no mistakes will be made. Patricia van den

Bremt thinks we should be focused on the avoidable harm, which I agree. She also talked

about a HARM (Hospital Admission related to medication) study. The aim of this study is to

identify the costs, the risk factors and the frequency and potential preventability. The results

are that there is still quite a lot of improvement possible and that a good communication is

important.

I think it is important to know why a drug is chosen and how to prescribe it, so the drug

would be used for the correct reasons and the preventable harm would reduce. And I agree

with Patricia van den Bremt that it is important to improve the medication safety.

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