Development of methods for analysis of synthetic ... · PDF fileDevelopment of methods for...

Universität Basel Philosophisch-Naturwissenschaftliche Fakultät Departement Pharmazeutische Wissenschaften

Institut für Pharmazeutische Biologie

Development of methods for analysis of synthetic adulterants in herbal

medicines by HPTLC

Diploma Thesis

Seraina Caprez

Supervision: Prof. Matthias Hamburger, Universität Basel

Dr. Eike Reich, CAMAG Muttenz Anne Schibli, CAMAG Muttenz

May - September 2005

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Danksagung Mein Dank geht an:

- Prof. Dr. Matthias Hamburger für die Leitung der Arbeit

- Die Firma CAMAG für den Arbeitsplatz und die Materialien

- Dr. Eike Reich und Anne Schibli für die Betreuung und die fachliche Unterstützung

- Valeria Widmer für die Hilfe bei der Informationssuche in den Weiten des Internets,

Daniel Handloser für die Hilfe bei Laborfragen und Katherine Gessler für das Tei-

len von Freund und Leid während der Arbeit

- Dr. Olivier Potterat für die Hilfe bei der HPLC-Analytik

- Dr. Hans-Ruedi Schmutz und Dr. Christelle Jablonski-Lorin für die Hilfe bei der

NMR-, IR-, und MS-Analytik

- Meine Familie, meine Freundinnen und alle, die mich während dieser Zeit unter-

stützt haben

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Table of contents Danksagung.................................................................................................................. 3

Abstract......................................................................................................................... 6

Zusammenfassung ....................................................................................................... 7

1. Introduction............................................................................................................ 9

1.1. Chinese herbal medicine............................................................................ 9

1.2. Aim of this Diploma Thesis....................................................................... 10

1.3. Thin-layer chromatography ...................................................................... 11

1.3.1. Principle ................................................................................................ 11

1.3.2. Influencing factors................................................................................. 12

1.3.3. Method development ............................................................................ 13

1.3.4. Quantitative evaluation ......................................................................... 15

1.3.5. Problems with natural products ............................................................ 15

2. Material and Methods.......................................................................................... 17

2.1. Material ..................................................................................................... 17

2.1.1. Samples ................................................................................................ 17

2.1.2. Standards.............................................................................................. 21

2.1.3. Plates .................................................................................................... 22

2.1.4. Chemicals ............................................................................................. 23

2.1.5. Equipment and accessories.................................................................. 24

2.1.6. Chemicals and Equipment for HPLC.................................................... 25

2.1.7. Chemicals and Equipment for NMR ..................................................... 25

2.2. Methods.................................................................................................... 26

2.2.1. Sample preparation .............................................................................. 26

2.2.2. Standard preparation ............................................................................ 27

2.2.3. Methods for HPTLC .............................................................................. 29

2.2.4. Methods for derivatization..................................................................... 32

2.2.5. Method for HPLC .................................................................................. 32

3. Results and Discussion ....................................................................................... 33

3.1. Method optimization for anti-impotency drugs ......................................... 33

3.1.1. Existing method and optimization......................................................... 33

3.1.2. Sample analysis.................................................................................... 34

3.1.3. Results from HPLC-analysis................................................................. 36

5

3.1.4. Reproducibility, influence of relative humidity and chamber saturation37

3.1.5. Quantification of Sildenafil .................................................................... 39

3.1.6. Discussion............................................................................................. 42

3.2. Method development for anti-rheumatic drugs ........................................ 43

3.2.1. Existing methods................................................................................... 43

3.2.2. Method development ............................................................................ 43

3.2.3. Sample analysis.................................................................................... 46

3.2.4. Results from NMR-, MS- and IR-analysis............................................. 49

3.2.5. Discussion............................................................................................. 50

3.3. Method development for anti-diabetic drugs............................................ 51

3.3.1. Existing method .................................................................................... 51

3.3.2. Method optimization.............................................................................. 51

3.3.2.1. Developing solvent ........................................................................ 51

3.3.2.2. Derivatization reagent.................................................................... 52

3.3.3. Sample analysis.................................................................................... 53

3.3.4. Discussion............................................................................................. 56

3.4. Final Discussion ....................................................................................... 57

4. List of Figures...................................................................................................... 59

5. Literature References.......................................................................................... 60

6. Annex .................................................................................................................. 62

6.1. List of Abbreviations ................................................................................. 62

6.2. LC/MS spectra of anti-impotency drugs ................................................... 64

6.3. NMR spectrum of anti-rheumatic drug ..................................................... 72

6.4. MS spectrum of anti-rheumatic drug ........................................................ 73

6.5. IR spectrum of anti-rheumatic drug.......................................................... 80

6

Abstract Traditional Chinese medicine (TCM) enjoys great popularity in western countries.

The distribution of TCM products has much increased and become a lucrative busi-

ness. The prospect of fast profit attracts dubious companies, which act only via the

internet. This market is nearly uncontrollable and implicates several risks. In general

the quality of such products is not ensured. Contamination with chemicals, heavy

metals, banned pesticides and microbes are a known problem in Asian herbal medi-

cines. But there are also cases of adulteration with synthetic agents.

The aim of this diploma thesis was to develop simple and rapid high performance

thin-layer chromatographic methods for the detection of synthetic adulterants in

herbal preparations.

In a first part method development and sample analyses for anti-impotency drugs are

discussed. Optimal solvent is tert-Butyl methyl ether, methanol, ammonia (20:2:1); as

solid phase silica gel plates are used.

The second part contains method development and sample analyses for anti-

rheumatic drugs. As solvent systems heptane, 2-propanol, acetic acid (15:5:1) for

non-steroidal anti-inflammatory drugs and dichloromethane, acetone (3:1) for corti-

costeroids are suitable. As solid phase silica gel plates are suitable.

The third part deals with the method development and sample evaluation for anti-

diabetic agents. Best separation is achieved on RP18W plates and 2-propanol, wa-

ter, acetic acid (10:20:5) as solvent system. Derivatization is effected with sulfuric

acid reagent.

With all three methods artificial and real samples were tested for synthetic adulter-

ants.

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Zusammenfassung Traditionelle chinesische Medizin (TCM) erfreut sich in westlichen Ländern als Teil

der Komplementärmedizin immer grösserer Beliebtheit. Der Vertrieb von Arzneimit-

teln nahm in den letzten Jahren massiv zu und ist zu einem lukrativen Markt ge-

wachsen. Die Aussicht auf schnellen Profit zieht aber auch viele dubiose Firmen, die

ihre Geschäfte mitunter nur über das Internet abwickeln, an. Dieser Markt ist sehr

schwer kontrollierbar und birgt einige Risiken. Die Qualität von solchen Arzneimitteln

ist im Allgemeinen nicht gewährleistet. Kontamination mit Chemikalien, Schwermetal-

len, verbotenen Pestiziden und Mikroben sind ein bekanntes Problem bei pflanzli-

chen Arzneimitteln asiatischer Herkunft. Immer wieder treten aber auch Fälle von

Verfälschungen mit synthetischen Wirkstoffen auf.

Ziel dieser Diplomarbeit war es, einfache dünnschichtchromatographische Methoden

zu entwickeln, um solche Verfälschungen rasch und zuverlässig nachweisen zu kön-

nen.

In einem ersten Teil wird eine Methodenentwicklung und Probenuntersuchung für

Potenzmittel besprochen. Optimales Laufmittel ist tert-Butylmethylether, Methanol,

Ammoniak (20:2:1), als feste Phase werden Silicagel-Platten verwendet.

Der zweite Teil enthält eine Methodenentwicklung und Probenanalyse für Antirheu-

matika. Als Fliessmittel eignet sich für Nicht-steroidale Antirheumatika Heptan,

Isopropanol, Essigsäure (15:5:1), für Corticosteroide Dichlormethan, Aceton (3:1).

Als feste Phase werden Silicagel-Platten eingesetzt.

Der dritte Teil befasst sich mit der Methodentwicklung und Probenauswertung für

antidiabetische Medikamente. Die beste Trennung von verschiedenen oral wirksa-

men Antidiabetika wird auf RP18W-Platten mit Isopropanol, Wasser, Essigsäure

(10:20:5) erreicht. Derivatisierung erfolgt mit Schwefelsäurereagens.

Mit allen drei Methoden wurden künstliche und echte Proben auf Verfälschungen mit

synthetischen Wirkstoffen untersucht.

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1. Introduction

1.1. Chinese herbal medicine

Pharmaceutical products from traditional Chinese medicine (TCM) contain one or

more active substances, all of which are derived wholly from plants, animals or min-

erals. The medicinal products or all of its active substances are described in the cur-

rent edition of “The Chinese Herbal Medicine Materia Medica” or an equivalent publi-

cation. TCM does not include any medicinal product that contains as an active sub-

stance any chemically defined, isolated constituents of plants, animals or minerals

[1], [2].

TCM is gaining popularity in western countries as a form of complementary and al-

ternative medicine (CAM). Very little is known concerning the reasons for the in-

creased popularity of CAM in developed countries. Untested assumptions include the

side-effects of synthetic drugs, general dissatisfaction of orthodox treatment, dis-

eases that can not be cured with conventional therapies and patients with chronic

illnesses demanding more attention from western practitioners.

However several problems may be associated with herbal medicinal products. They

may be contaminated with excessive or banned pesticides, microbial contaminants,

heavy metals and chemical toxins [3]. Whereas problems relating to the toxicity of

their herbal ingredients have been repeatedly reviewed, other important safety issues

have been neglected for a long time. One such topic is the possibility of adulteration

of TCM products with conventional drugs [4].

Recently more and more attention is paid to this problem. Many reports about adul-

terations and adverse reactions due to synthetic ingredients have been elaborated

[3-10]. Most common adulterants are all sorts of pain killers, Cortisone and its deriva-

tives, anti-allergic substances, oral anti-diabetic agents and lifestyle medicines as

anorexic drugs and anti-impotency drugs. Problems resulting from this kind of adul-

terants are not negligible.

People’s Republic of China is in the endeavor to improve manufacturing conditions

and to apply actual Good Manufacturing Practice (GMP) guidelines to comply with

the expected quality standard of the growing western markets. Quality control is in-

creased and inspection reinforced.

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However the opposite side is not inactive either. Inventive producer try to defy ana-

lytical control by decreasing the quantity of the ingredient below the limit of detection.

Other manufacturers add structure-modified compounds that are hard to determine.

Among others, four anti-impotency drug analogues have been determined and their

chemical structures have been elucidated. As these substances are synthesized un-

der dubiously conditions and effects and toxicity have not been tested, they may ex-

ceed Sildenafil’s risks and complications [11].

Furthermore many companies sell their products through the internet, which can

hardly be supervised and it will be a big challenge to governmental controlling bod-

ies.

1.2. Aim of this Diploma Thesis

TCM is gaining popularity in western countries. Distribution of TCM products has be-

come a lucrative business. Besides reputable manufacturers a multitude of dubious

internet shops try to get their piece of the action. Drugs purchased via the internet

are often labeled only in Chinese. Specifications of ingredients are rarely declared.

Where effectiveness is insufficient impostors adulterate so called purely herbal TCM

medicines with chemical active ingredients.

So far most analytical methods for the detection of synthetic adulterants in herbal

medicines were developed for HPLC/MS.

The aim of this diploma thesis was to develop simple and rapid HPTLC methods for

the detection of synthetic adulterants in herbal preparations as a contribution to the

pharmaceutical drug safety.

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1.3. Thin-layer chromatography

Thin-layer chromatography is of great importance for the analysis of natural products.

A large number of complex samples can rapidly be analyzed under the same condi-

tions. Analysis and sample preparation time is generally short; many samples can

easily be compared side by side on the same plate. This advantage is especially

convenient for the analysis of fingerprints. A broad spectrum of constituents can thus

be detected and described without the need to know the chemical nature of each

zone of the chromatogram. Results are not only reported as peak data but can also

easily be presented and communicated as images [12].

The availability of modern, usually computer-controlled, equipment has unlocked the

full power of the method and opened new fields of qualitative and quantitative appli-

cations of planar chromatography. Today’s instruments, such as automatic applica-

tion devices, sophisticated developing devices, scanning densitometers, and video

documentation systems, have complemented the inherent advantages of TLC with

increased reliability, better sensitivity, and improved precision and accuracy of the

analytical result. Even tough all modern TLC equipments can also be used with con-

ventional plates, it should be understood that only with HPTLC plates is the maxi-

mum performance achieved and all advantages of the technique realized. [13].

However HPTLC is not always a suitable method. Other chromatographic techniques

(GC, HPLC) with a higher resolving power combined with a detector (MS, IR, NMR)

are still best choice for many problems.

1.3.1. Principle

Thin-layer chromatography is an analytical technique for the separation of mixtures. It

involves passing a sample (the analyte) in the mobile phase through the stationary

phase. Usually, each component has a characteristic migration distance that can be

used to identify it and thus the composition of the original mixture. Depending on the

plate material different mechanisms like absorption, partition, ion exchange and com-

plexation have an impact.

TLC is a flexible, versatile, and economical process in which the various stages

(sample preparation, application, chromatogram development and evaluation) are

12

carried out independently. The advantages of this off-line arrangement as compared

to an on-line process, such as column high performance liquid chromatography

(HPLC), have been outlined and include the following (according to [14]):

1. Availability of a great range of stationary phases with unique selectivities for

mixture components

2. Ability to choose solvents for the mobile phase is not restricted by low UV

transparency or the need for ultra-high purity

3. Repetition of densitometric evaluation can be achieved under different condi-

tions without repeating the chromatography in order to optimize quantification

since all sample fractions are stored on the plate

4. High sample throughput since many samples can be chromatographed simul-

taneously

5. Minimal cost of solvent purchase and disposal since the required amount of

mobile phase per sample is small

6. Specific components can be optimized selectively

7. No cross contamination as plates are used only once

1.3.2. Influencing factors

Influencing factors in HPTLC are various. As HPTLC-Systems are open systems and

therefore sensitive to variations, reproducibility is only warranted if influencing factors

are kept constant. A standardized methodology allows reproducible results. Never-

theless, some parameters are tested in the following.

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1.3.3. Method development

Figure 1-1 Method development schema

Figure 1-1 shows a schematic overview of a method development. At the beginning

sample preparation, chromatographic system and stationary phase have to be de-

fined. Then the mobile phase can be composed and the detection optimized.

For the mobile phase, it is advisable to search the literature for methods for similar

substances and to test them. If the existing methods do not yield satisfying results,

the CAMAG optimization scheme (see figure 1-2) for method development can be

adopted.

Figure 1-2 CAMAG optimization scheme

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Chamber At the beginning of an optimization the chamber should be determined. In this work

saturated twin trough chambers according to the HPTLC SOP of CAMAG were used.

They permit a high reproducibility and reduce the risk of solvent demixing.

Stationary phase Silica gel is the most commonly used plate material. The porous texture allows an

excellent separation for a majority of substances.

Chemically modified plates with RP18-, diol-, cyano- or amino-phases may be useful

if silica gel does not yield the required separation.

Developing solvent In a first step chromatograms are developed with neat solvents of every selectivity

group (cf. table 1-1). Subsequently solvent strength is decreased with hexane or in-

creased with methanol or water. Afterwards mixtures of best solvents are tested and

modified with acids or bases if needed. With variations of the proportions best results

can be expected.

Table 1-1 Selectivity groups (according to Snyder [15])

I Aliphatic ethers, trialkylamines, trialkylphosphates II Aliphatic alcohols III Pyridine, THF, DMSO, DMF, diethylene glycol IV Benzyl alcohol, ethylene glycol, acetic acid, formamide V Dichloromethane, 1,2-dichloroethane VI Ketones, esters, dioxane, nitriles VII Aromatic (halogenated) hydrocarbons, nitro comp. VIII Chloroform, water, fluoroalcohols, m- cresol

Detection Visualization is accomplished under UV at 254 nm (extinction of the indicator’s fluo-

rescence), under UV at 366 nm (fluorescence of substance) or under white light.

15

1.3.4. Quantitative evaluation

Quantitative evaluation is possible with densitometric analysis of the chromatogram.

During evaluation with a modern densitometer the separation tracks are scanned

with a light beam of defined size. The densitometer is, therefore, commonly referred

to as scanner.

A photo sensor in the densitometer measures diffusely reflected light. The difference

between the optical signal from the sample-free background and that from a sample

zone is measured. The signal must be correlated with those from different amounts

of calibration standards chromatographed on the same plate [16].

To define the optimal wavelength for measurement, a spectrum of the designated

substance is taken.

1.3.5. Problems with natural products

Asian herbal medicines generally consist of a combination of different natural prod-

ucts. Samples are complex mixtures of compounds, active principles are often un-

known. This makes analytical investigations rather difficult. Furthermore RF values

and UV spectra provide only limited information about the sample constitution. Reli-

able analyses are therefore not always evident.

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2. Material and Methods

2.1. Material

2.1.1. Samples

Anti-impotency drugs S1921 America Penis God

Declared ingredients Thiocyanic acid propylester, Methyl 1605, Potassium sorbate,

Asparagus root, Ammoniac Phenylalanine methylester

Manufacturer American Omena Group LLC

Source China, received from Korean custom

This sample was analyzed with HPLC at the Korean custom and was found to con-

tain Sildenafil [11].

S1922 Men Treasure

Declared ingredients unknown

Manufacturer unknown

Source China, received from Korean custom


tain Sildenafil and Homosildenafil [11].

S1923 America Treasure Declared ingredients unknown


Source Taiwan, received from Korean custom


tain Sildenafil [11].

18

S1924 GOLDEN 1-HIWELL Declared ingredients Changbai Mountains wild Ginseng, Piper longumi, essence of

actinolite, essence of Epimedium sagittanum

Manufacturer Swiss Treasure Health Supplement Ltd.

Source purchased in Hong-Kong

S1925 PowerX

Declared ingredients Maca, Tongkat Ali

Manufacturer First Choice Holdings Ltd.


S1926 Super King

Declared ingredients Herba Epimedii, Royal Jelly, Glycyrrhiza Uralensis Fisch

Manufacturer China Shineway Pharmaceutical Ltd.


Anti-rheumatic drugs S2101 Artificial sample Mix 1

Ingredients 1 g ASA, 0.2 g Caffeine, 0.8 g Acetaminophen, 2 g TCM

plants (Angelica, Rhemannia, Ginseng root)

S2102 Artificial sample Mix 2

Ingredients 8 mg Dexamethasone (2 tablets), 10 mg Prednisone (2 tab-

lets), 400 mg Ibuprofen (2 tablets), 3.58 g TCM plants (Angel-

ica, Rhemannia, Ginseng root)


Ingredients 0.2 g Propylphenzone, 0.5 g Mefenamic acid (2 capsules),

2.8 g TCM plants (Angelica, Rhemannia, Ginseng root)

19

S2110 Panosin (traditional Korean pain killer)

Declared ingredients Ponciri Fructus, Paeoniae Radix, Platycodi Radix, Zingiberis

Rhizoma, Zizyphi Fructus, Glycyrrhizae Radix

Manufacturer Jung Woo Pharmaceutical Co.

Source purchased in Korea

S2111 Maroicin (traditional Korean painkiller)

Declared ingredients among others Paeoniae Radix, Zizyphi Fructus, Glycyrrhiz-

hae Radix, Hovenia dulcis



S2112 Yorosin (traditional Korean pain killer)

Declared ingredients Rehmanniae Radix, Angelicae gigantis Radix, Akebiae Cau-

lis, Scutellariae Radix, Alismatis Rhizoma, Plantaginis Se-

men, Glycyrrhizae Radix, Gardeniae Fructus, Gentianae

Scabrae Radix, Cinnamomi Cortex, Zingiberis Rhizoma

Crudus, Rhei Rhizoma, Forsythiae Fructus



S2113 Iszpen (combined Korean pain killer)

Declared ingredients Pulvis Peoniae Radicis, Pulvis Glycyrrhizae Radicis, Acet-

aminophen, Ethenzamide, Caffeine



20

S2116 Comdrugcide Pain-killer decoction

Declared ingredients Cortex Cinnamomi, Radix Codonopsis, Cortex Eucommiae,

Radix Paeoniae Alba, Rhizoma Cyperi, Semen Ziziphi Spino-

saei, Stephania Cepharantha Hayatai, Herba Asarii

Manufacturer Comdrugcide Medical Co. Hong Kong

Source purchased via internet

Anti-diabetic drugs S2107 Artificial sample Mix 4

Ingredients 2.5 mg Glibenclamide (1/2 tablet Daonil®), 3 g TCM plants

(Rhemannia, Schisandra Extract, Astragalus Root)


Ingredients 500 mg Metformin (1tablet), 2.5 g TCM plants (Rhemannia,

Schisandra Extract, Astragalus Root)

S2109 D-Abin (traditional Korean anti-diabetic drug)

Declared ingredients Liriope Tuber, Ginseng Radix Alba, Puerariae Radix, Reh-

manniae Radix Preparata, Anemarrhenae Rhizoma, Tricho-

santhis Radix, Hoelen, Schizandrae Fructus, Glycyrrhizae

Radix, Araliae Radicis Cortex



S2117 Xiaoke

Declared ingredients Radix Pueriariae, Radix Rhemanniae, Radix Astragali, Radix

Trichosanthis, Stylus Zeae Mayadis, Fructus Schisandrae

Sphenantherae, Rhizoma Dioscoreae, Glibenclamide

Manufacturer Guangzhou Zhongyi Pharmaceutical Co.


21

S2118 Ye Tianshi Declared ingredients Astragali, Pollen, Panax quinquefolium, Dried Rhemannia

Root, Anoderma Lucidum, Pueraria Root, Dioscoream,

Schisaema, Cordyceps

Manufacturer Hong Kong Shun Chong Trading Co.


S2159 Plant matrix Ingredients Rhemannia, Schisandra, Astragalus (3:1:1)

2.1.2. Standards

Anti-impotency drugs Standard No. Standard description Lot R1592 Yohimbin 5mg Hänseler 2003070301

R1593 Viagra® (Sildenafil) 25 mg Pfizer 3105212

R1594 Cialis® (Tadalafil) 10 mg Lilly A059503

R1595 Levitra® (Vardenafil) 5 mg Bayer BXB9XL1

R1927 Hongdenafil (solution 413 ppm) Korean custom

R1928 Aminotadalafil Korean custom

(solution conc. unknown)

R1929 Tadalafil (solution 402 ppm) Korean custom

R1930 Homosildenafil (solution 397 ppm) Korean custom

R1931 Hydroxy-homosildenafil Korean custom

(solution 396 ppm)

R1932 Sildenafil (solution 382 ppm) Korean custom

R1933 Vardenafil (solution 401 ppm) Korean custom

22

Anti-rheumatic drugs

Standard No. Standard description Lot R2064 Dexamethasone tablet 4 mg Galepharm 0105

R2065 Prednisone tablet 5 mg Galepharm 0604

R2066 Prednisolone tablet 5 mg Galepharm 0902

R2073 Propylphenazone PhEur Hänseler 6-7426-1

R2074 Acetylsalicylic acid Fluka 259066 391

R2075 Acetaminophen (Paracetamol) Serva 10035

R2084 Caffeine Merck 1.02584.0250

R2085 Voltaren Dolo® (Diclofenac 12.5 mg) Novartis unknown

R2086 Ponstan® (Mefenamic acid 250 mg) Parke Davis 0195083

R2087 I-2® (Ibuprofen 200 mg) Equate AE0110

Anti-diabetic drugs

Standard No. Standard description Lot R2062 Metformin-Mepha® 500 Mepha 480468

R2063 Daonil® (Glibenclamide 5 mg) Aventis 40D102

2.1.3. Plates

Specification Manufacturer Lot HPTLC glass 10X10, Si 60 F254 Merck OB464935

OB412660

HPTLC glass 20X10, Si 60 F254 Merck 940348957

OB524408

OB526793

OB302430

HPTLC glass 20X10, RP18 F254s Merck 11789100

HPTLC glass 10X10, RP18 WF254s Merck 22859929

OB102016

23

2.1.4. Chemicals

Specification Manufactuerer Lot. No. Acetic acid 100% p.a. Merck K27853063

Acetone p.a. Acros A019663101

Acetonitrile ISO 9001 Riedel-de Haën 5265A

Ammonia 28% Fluka 407908/1 41500

Anisaldehyde Fluka 10440

tert-Butyl methyl ether p.a. Merck 720195

Chloroform p.a. Acros A09834601

Cyclohexane p.a. Merck K10066566 815

p-Chloranil Merck 802361

Dichloromethane p.a. Acros A020544801

Ethanol p.a. Merck K33957583

Ethyl acetate p.a. Merck K33137923415

Iron(III)chloride hexahydrate Merck 1.03943.0250

Formic acid 99% Acros A019883701

n-Hexane p.a. Merck L054367 237

n-Heptane p.a. Merck K25693579 841

Hydochloric acid 37% p.a. Merck K31897217

Hydrogen peroxide extra pure Merck K33052400 421

Methanol p.a. Acros A0205287001

Potassium hydrogen phosphate Pharmacy Apogast 2002.04.0197

Potassium hexacyanoferrate(III) Merck 4973

2-Propanol p.a. Merck K32940634 407

Sodium hydroxide Merck 6498

Sulfuric acid 96% p.a. Merck -

Tetrahydrofuran p.a. Merck K29252431 120

Toluene p.a. Merck K33553125 432

24

2.1.5. Equipment and accessories

Specification Manufacturer Serial No. AccuBOND® C18 cartridges for SPE J&W Scientific 13110

ADC Light µP controlled CAMAG Prototype

Analytical balance AG245 Mettler Toledo 1114402254

Automatic Development Chamber 2 CAMAG 120424

Automatic TLC Sampler 4 CAMAG 061104

Centrifuge EBA 21 Hettich 0000799-01-00

Chromatogram Immersion Device III CAMAG -

Digital camera G5 Canon -

Hair dryer Starline 301 Solis -

Linomat 5 CAMAG 080224

Oven for plate drying, Thermocenter Salvis -

Reprostar 3 CAMAG 070705

Rotary Evaporator Büchi -

TLC Plate Heater III CAMAG 981109

TLC Scanner 3 CAMAG 041118

TLC Spray Cabinet II & Spray device CAMAG 10101

Twin Trough Chamber 20x10 cm / 10x10 cm CAMAG -

Ultrasonic bath TPC-25 Telsonic 2003043

SPE-Manifold, VacElut AT 6000 Analytichem Int. 1814

winCATS Software CAMAG Version 1.3.3

25

2.1.6. Chemicals and Equipment for HPLC

Equipment and accessories Specification Manufacturer Serial No. Automatic HPLC sampler: Liquid Handler Gilson -

Column Nucleodur C18 Gravity, 5 µm Macherey-Nagel 5051018

HPLC 1100 Series: Agilent

Degasser JP40717412

Binary Pump DE40915968

Detector DE40540853

Mass spectrometer Esquire 3000 plus Brukner 73700.01085

Software Histar -

Chemicals for HPLC

Chemical Manufacturer Lot. No. Acetonitril Multisolvent Scharlau Chemie -

Formic acid 98% p.a. Fluka -

2.1.7. Chemicals and Equipment for NMR

Equipment Specification Manufacturer Serial No. NMR B-ACS-60 Brukner -

Chemicals Chemical Manufacturer Lot. No. MeOD - -

26

2.2. Methods

Samples were dissolved either in acidic water or in methanol in ultrasonic bath for 30

minutes.

Dilutions were made with the extraction solvent. Percentages at the end of the sam-

ple indicate the factor of the dilution.

2.2.1. Sample preparation

Method 1 The sample (one tablet or capsule) was dissolved in 50 mL acidic water (pH 4 ad-

justed with acetic acid) in ultrasonic bath for 30 minutes. The suspension was centri-

fuged and the supernatant was used. S1924-01, S1925-01, S1926-01

Method 2 The sample (one tablet or capsule) was dissolved in 10 mL acidic water (pH 4 ad-

justed with acetic acid) in ultrasonic bath for 30 minutes. The suspension was centri-

fuged and the supernatant was used. S1921-01, S1922-01, S1923-01, R1592-01

Method 3 The sample (recommended single dose) was pounded and dissolved in 10 mL

MeOH in ultrasonic bath for 30 minutes. The suspension was centrifuged and the

supernatant was used.

S2110-01, S2111-01, S2112-01, S2113-01, S2109-01, S2117-01-02, S2118-01

Method 4 0.5 g of the sample were dissolved in 10 mL MeOH in ultrasonic bath for 30 minutes.

The suspension was centrifuged and the supernatant was used.

S2101-02, S2102-02, S2103-02

27

Method 5 0.3 g of the sample were dissolved in 10 mL MeOH in ultrasonic bath for 30 minutes.

The suspension was centrifuged and the supernatant was used.

S2107-01, S2108-01

Method 6

6 mL of the dissolved sample were cleaned up with SPE over a C18 cartridge. The

filtrate was discarded and the sample was eluted with 0.3 mL MeOH.

S1924-01-SPE, S1925-01-SPE, S1926-01-SPE

Method 7 2 mL of the liquid sample were cleaned up with SPE over a C18 cartridge. The filtrate

was discarded and the sample was eluted with 0.3 mL MeOH.

S2116-SPE

2.2.2. Standard preparation

Active ingredients were dissolved in methanol.

Drugs standards were dissolved either in methanol or in acidified methanol in ultra-

sonic bath for 30 minutes.

Dilutions were made with the extraction solvent. Percentages at the end of the sam-

ple indicate the factor of the dilution.

Method 8 for anti-impotency drugs (according to method developed by CAMAG) One tablet was dissolved in 10 mL MeOH-HCl 37% (9:1) in ultrasonic bath for 30

minutes. The suspension was centrifuged and the supernatant was used.

R1593-01, R1594-01, R1595-01

Method 9 for liquid Sildenafil and analogues 100 µL of the liquid standard were dissolved in 900 µL MeOH.

R1927-01, R1928-01, R1929-01, R1930-01, R1931-01, R1932-01, R1933-01

28

Method 10 for active ingredients of NSAIDs 50 mg of the standard were dissolved in 10 mL MeOH.

R2073-01, R2074-01, R2075-01, R2084-01

Method 11 for anti-rheumatic and anti-diabetic drugs

One tablet was dissolved in 10 mL MeOH in ultrasonic bath for 30 minutes. The sus-

pension was centrifuged and the supernatant was used.

R2062-02-02, R2063-02-02, R2064-01, R2065-01, R2066-01, R2085-01, R2086-01,

R2087-01

29

2.2.3. Methods for HPTLC

Method A for anti-impotency drugs Stationary phase HPTLC glass 10X10 cm / 20x10 cm Si 60 F254 Merck

Chamber TTC 10x10cm / 20x10 cm

Application according to the concentration 2 – 20 µL of standards

and samples applied as 8 mm bands, at least 2 mm

apart, 8 mm from the lower edge of the plate

Developing solvent 5 mL / 10 mL; TBME, MeOH, NH3 (20:2:1), upper phase

Saturation 20 min. with filter paper

Developing distance 62 mm from application position

Visualization under UV at 254 nm and 366 nm

Detection spectral detection 200 – 400 nm with Scanner 3

Method B for NSAID Stationary phase HPTLC glass 10X10 cm / 20x10 cm Si 60 F254 Merck





Developing solvent 5 mL / 10 mL; Heptane, 2-propanol, HAc (15:5:1)





30

Method C for Corticosteroids (according to [17]) Stationary phase HPTLC glass 10X10 cm / 20x10 cm Si 60 F254 Merck





Developing solvent 5 mL / 10 mL; DCM, Acetone (3:1)





Method D for anti-diabetic drugs Stationary phase HPTLC glass 10X10 cm, RP18 WF254s Merck

Chamber TTC 10x10cm




Developing solvent 5 mL





31

Method E for preparative plates of anti-impotency drugs Stationary phase HPTLC glass 20x10 cm Si 60 F254 Merck

Chamber TTC 20x10 cm

Application 500 µL as a 180 mm band, 8 mm from the lower edge of

the plate

Developing solvent 10 mL; TBME, MeOH, NH3 (20:2:1)





The designated zone was scraped off and the substance eluted with 1 mL MeOH.

Samples were labeled with the appendix –prep.

Method F for preparative plates of anti-rheumatic drugs Stationary phase HPTLC glass 20x10 cm Si 60 F254 Merck

Chamber TTC 20x10 cm

Application 500 µL as a 180 mm band, 8 mm from the lower edge of

the plate

Developing solvent 10 mL; Heptane, 2-propanol, HAc (15:5:1)





The designated zone was scraped off and the substance eluted with 1 mL MeOH.

Samples were labeled with the appendix –prep.

32

2.2.4. Methods for derivatization

Method I for p-Chloranil-Reagent [18] Preparation 0.5 g p-chloranil were dissolved in 90 mL ethyl acetate

and cautiously made up to 100 mL with sulfuric acid

96%.

Reagent use dipping

Heating 5 minutes at 100°C

Detection under WRT and UV at 366 nm

Method II for Anisaldehyde-Reagent R [19] Preparation 0.5 mL anisaldehyde R, 10 mL acetic acid 99% R,

85 mL methanol R and 10 mL sulphuric acid R were

mixed in chronological order.

Reagent use dipping



Method III for H2SO4-Reagent Preparation 10 mL H2SO4 96% were made up to 100 mL with

methanol

Reagent use dipping



2.2.5. Method for HPLC

Mobile phase Water / formic acid 1%, Acetonitrile

Gradient from 90:10 to 0:100 within 20 minutes

Flow rate 0.4 mL / min

Column temperature 35°C

Injection volume 5 – 20 µL depending on the concentration

33

3. Results and Discussion

3.1. Method optimization for anti-impotency drugs

3.1.1. Existing method and optimization

Method A was used, but the solvent (TBME, MeOH, Ammonia 28% 20:1:1, upper

phase) from an existing method developed by CAMAG was tested. As the standards

could not be separated very well (see figure 3-1), the method was optimized.

The proportion of methanol in the developing solvent was increased. Developing sol-

vent: TBME, MeOH, Ammonia 28% (20:2:1), upper phase. The solvent system was

found to be appropriate (cf. figure 3-2).

Figure 3-1 Anti-impotency drugs, standards and samples

Figure 3-2 Anti-impotency drugs, standards and samples; optimized solvent

1a: R1592-01 Yohimbin, 1b: R1593-01 (Viagra®, Sildenafil), 2: R1594-01 (Cialis®, Tadalafil), 3: R1595-01 (Levita®, Vardenafil), 4: S1924-01, 5: S1925-01, 6: S1926-01

The standards were applied on track 1 to 3. Multiple bands are visible, so it can be

concluded that the standards are not stable. As in further tests standard solutions

have been used, no stability tests were performed.

On track 4 to 6 three different samples were applied. Light blue spots from the sam-

ple matrix remain at the application position. Samples should be cleaned up with

SPE to remove the matrix and to concentrate the solution.

1b 2 3 4 5 6 1a 1b 2 3 4 5 6

34

3.1.2. Sample analysis

Samples S1921, S1922, S1923, S1924, S1925 and S1926 were analyzed with

method A. Plates were evaluated visually and with UV spectra comparison (cp. fig-

ures 3-3 to 3-8).

Spectra were measured in a range from 200 nm to 400 nm.

Figure 3-3 Standard solutions and samples from Korean custom

1: R1927-01 (Hongdenafil), 2: R1928-01 (Aminotadalafil), 3: R1929-01 (Tadalafil), 4: R1930-01 (Ho-mosildenafil), 5: R1931-01 (Hydroxyhomosildenafil), 6: R1932-01 (Sildenafil), 7: R1933-01 (Varde-nafil), 8: Sample S1921-01, 9: Sample S1922-01, 10: Sample S1923-01

Figure 3-4 UV spectrum of standard Sildenafil and samples from Korean custom

Figure 3-5 UV spectrum of standard Homosildenafil and sample S1922

Figure 3-3 shows the standard solutions and the samples from the Korean custom.

On track 3, 6 and 7 three active ingredients from registered anti-impotency drugs

were applied. Track 1, 2, 4 and 5 contain four chemically modified illegal substances.

On track 8 to 10 three samples were analyzed.

1 2 3 4 5 6 7 8 9 10

35

Standards R1927 Hongdenafil and R1928 Aminotadalfil are not stable in solution.

Two bands with the same UV spectrum appear.

Standard Sildenafil on track 6 appears at a RF value of 0.15. On tracks 8 to 10 on the

same height a bright blue band can be seen. All of these spots have the same UV

spectrum as the reference (cp. figure 3-4) and can therefore be identified as Silde-

nafil. Additionally sample S1922 on track 9 shows a band at the position of Homosil-

denafil. Spectra comparison (cf. figure 3-5) allows a reliable identification of the pres-

ence of Homosildenafil in the sample.

Figure 3-6 Standard solutions and samples purchased in Hong Kong

1: R1927-01 (Hongdenafil), 2: R1928-01 (Aminotadalafil), 3: R1929-01 (Tadalafil), 4: R1930-01 (Ho-mosildenafil), 5: R1931-01 (Hydroxyhomosildenafil), 6: R1932-01 (Sildenafil), 7: R1933-01 (Varde-nafil), 8: Sample S1924-01-SPE, 9: Sample S1925-01-SPE, 10: Sample S1926-01-SPE

Figure 3-7 UV spectrum of standard Silde-nafil and analogues and sample S1925

Figure 3-8 UV spectrum of standard Silde-nafil and analogues and sample S1926

1 2 3 4 5 6 7 8 9 10

36

Figure 3-6 shows the Standard solutions and samples purchased in Hong Kong.

Samples S1924, S1925 and S1926 were rather difficult to evaluate because of the

complex composition. The UV spectrum of each spot was compared to the spectra of

the reference substances. Zones with a similar spectrum were examined more

closely.

3.1.3. Results from HPLC-analysis

The zone at RF 0.39 of sample S1926 with a similar UV spectrum to Sildenafil was

isolated with method E and analyzed with HPLC / MS. Sample S1925 was analyzed

with HPLC / MS (method see chapter 2.2.5.).

Standard Sildenafil was measured as reference. Sample S1921-01 was analyzed to

verify the accuracy of the HPLC-method. Samples S1925-01-SPE and S1926-01-

SPE-Prep were examined.

Results are displayed in Annex 6.2.

For comparison molecular weights of Sildenafil and analogues are displayed in table

3-1 the ones of the analyzed samples in table 3-2

Table 3-1 Molecular weight of Sildenafil and analogues

Substance Molecular weight Sildenafil 474 Homosildenafil 488 Hydroxyhomosildenafil 504 Hongdenafil 466 Tadalafil 389 Aminotadalafil 390 Vardenafil 488

Table 3-2 Measured molecular weights of samples

Sample Molecular weight

S1925-01-SPE Peak 1 300 S1925-01-SPE Peak 2 260 S1925-01-SPE Peak 3 216 S1925-01-SPE Peak 4 246 S1925-01-SPE Peak 5 270 S1925-01-SPE Peak 6 244 S1926-01-SPE-Prep Peak 1 256 S1926-01-SPE-Prep Peak 2 266

In sample S1921, Sildenafil could be detected. Mass and fractions corresponded to

the reference. The peak at minute 1 is probably generated by the matrix.

The HPLC-method was found to be adequate.

37

In sample S1925-01-SPE no measured molecular weight corresponded to one of the

references. As detected masses are much lower than those of the references, a so

far unknown illegal analogue could be excluded.

In sample S1926-01-SPE-Prep two peaks could be detected with HPLC. Conse-

quently the sample was not as pure as presumed. The designated compound ap-

peared at minute 10.2. Its molecular weight is 256 which is not within the range of the

references. As in sample S1925, it is very unlikely, that the isolated substance is a

structure-modified anti-impotency substance.

3.1.4. Reproducibility, influence of relative humidity and chamber saturation

During sample analysis, it was found that RF values varied from plate to plate. There-

fore different influencing factors were examined.

Relative humidity To check the influence of the relative humidity on the RF values plates with samples

S1921-01-50%, S1922-01 and S1923-01-50% were developed in the ADC at two

different relative humidities. 6 plates with 15 tracks each (each sample 5 times, ap-

plied alternately) were measured. Within one plate, the RF values were very constant.

A comparison between the plates is shown in table 3-3.

Table 3-3 RF values of Homosildenafil and Sildenafil at different relative humidities

Relative humidity 47% Average value Deviation Plate 1 Plate 2 Plate 3 Homosildenafil 0.20 0.20 0.20 0.20 0.00 Sildenafil 0.09 0.10 0.09 0.09 0.01 Relative humidity 70% Average value Deviation Plate 4 Plate 5 Plate 6 Homosildenafil 0.32 0.33 0.34 0.33 0.01 Sildenafil 0.21 0.22 0.25 0.23 0.02

38

RF values rose when relative humidity was increased. If the relative humidity was

controlled by ADC, RF values remained constant. The results at a humidity of 47%

were more stable than at a humidity of 70%. To ensure the reproducibility, relative

humidity should be kept constant.

Chamber saturation

Chambers were always saturated for 20 minutes. But as the highly volatile Ammonia

is in the solvent system, slight differences in plate handling might cause changes in

the gas phase and therefore influence chromatography. However systematic tests

were not performed. As RF values did not change on plates developed with the ADC,

the use of this device is recommendable.

39

3.1.5. Quantification of Sildenafil

For the quantification of Sildenafil method A was used. Silica gel plates were pre-

washed with methanol.

From the UV spectrum of the compound the absorption maximum was determined at

wavelength 300 nm. Thus quantification test were performed at that wavelength.

The results were not reproducible. Measurements varied within one plate and be-

tween different plates. Test with different application modes did not improve the

situation. Results are displayed in tables 3-4 and 3-5 and figure 3-9.

On plate 1 different volumes were applied blockwise. On plates 2 to 4 different vol-

umes and concentrations respectively were distributed over the plate to minimize the

influence of varieties in between one plate. On plate 2 the same volume of different

concentrations was applied. On plate 4 syringe was filled twice before application to

prevent a dilution of the sample with the rinsing solution.

On plates 2 to 4 amounts tend to increase from one edge of the plate to the other.

Furthermore amounts calculated over height and over area vary slightly. This is also

visible on the displayed calibration curve on figure 3-9.

It is remarkable, that measured amounts decrease with increasing application vol-

ume.

If outliers are removed, fluctuations are slightly reduced but variations between plates

persist.

Variations between different plates are shown in table 3-5. No regular pattern is ob-

servable. Values do not decrease or augment with the time.

40

Table 3-4 Variation in quantification of sample S1921 Height Area Amount in appli-

cated volume (ng) Calculated average sample content (mg )

CV % Amount in appli-cated volume (ng)

Calculated average sample content (mg )

CV %

Plate 1 159.10 156.15138.80 137.79145.16 142.97

2 µL S1921-01-2% 142.34

36.59

6.08

150.07

36.69

4.48

159.28 211.80168.72 280.10156.33 225.73

3 µL S1921-01-2% 146.21

36.03

7.52

210.87

36.52

4.13

219.71 218.50203.85 199.52230.47 226.51

4 µL S1921-01-2% 260.49

28.55

10.45

254.52

28.20

10.16

Plate 2

102.55 107.9593.52 90.8687.02 84.40

2 µL S1921-01-2% 88.96

23.25

7.44

87.40

23.16

11.37

185.30 180.33173.37 198.00160.93 154.98

2 µL S1921-01-3% 160.83

28.35

6.89

159.35

27.61

6.75

295.42 287.41273.36 267.68256.05 258.83

2 µL S1921-01-5% 257.46

27.06

6.78

267.12

27.03

4.49

Plate 3

86.86 85.98- -

81.49 78.42

2 µL S1921-01-2% 101.11

21.27

14.77

100.13

22.04

20.00

161.68 165.95147.32 150.98165.97 168.37

4 µL S1921-01-2% 189.41

20.76

10.52

192.18

21.17

10.06

230.89 239.33223.99 238.33243.62 254.39

6 µL S1921-01-2% 270.65

20.19

8.50

273.51

20.95

6.55

Plate 4

143.45 141.26147.83 149.48149.17 149.04

2 µL S1921-01-2% 152.13

37.04

2.44

152.48

37.02

3.23

209.15 207.41212.26 208.57219.83 218.89

3 µL S1921-01-2% 219.72

35.87

2.50

218.28

35.55

2.88

257.65 254.84244.72 244.55257.49 257.47

4 µL S1921-01-2% 271.30

32.22

4.21

251.57

32.14

4.33

41

Table 3-5 Variation in quantification of sample S1921 between different plates

Height Area Calculated average sample

content (mg) Deviation (mg) Calculated average

sample content (mg) Deviation (mg)

Plate 1 33.72 5.69 33.80 4.85 Plate 2 26.22 2.65 25.93 2.42 Plate 3 21.02 0.54 21.39 0.58 Plate 4 35.04 2.51 34.90 2.50 Average all plates

29.00 6.59 29.01 6.46

Figure 3-9 Calibration curve of Sildenafil and measurements of sample S1921

42

3.1.6. Discussion

The solvent system TBME, MeOH, Ammonia (20:2:1) was found to yield best separa-

tion (method A). To ensure unchanging RF values, influencing factors such as cham-

ber saturation, relative humidity and temperature should be kept constant.

In samples S1921, S1922 and S1923 the presence of Sildenafil and Homosildenafil

respectively, could be proven.

In sample S1924 no synthetic adulterant could be detected. The complexity of sam-

ples S1925 and S1926 made the evaluation difficult. Zones with UV spectra similar to

known compounds were analyzed with HPLC/MS. No indications for the presence of

Sildenafil or analogues could be found.

The quantification of Sildenafil and Homosildenafil proved to be difficult. Problems

with high variations within one plate and between different plates could not be solved.

However it is conceivable, that the instable reference solution may have contributed

to the varying results. Quantifications should be performed with reference substance

in solid state.

43

3.2. Method development for anti-rheumatic drugs

For separation of non-steroidal anti-inflammatory drugs (NSAIDs) various methods

are described in literature, but none of them allows the simultaneous detection of dif-

ferent types of anti-rheumatic drugs (mainly NSAIDs and corticosteroids).

3.2.1. Existing methods

Different solvent systems described in literature were tested.

1) Tol, MeOH, NH3 (15:5:0.05) [20]

2) Cyclohexane, CHCl3, HAc (10:1:1) [21]

3) Tol, Acetone (8:2) (CAMAG method for analgetics)

4) DCM, EtOAc, Cyclohexane, 2-propanol, HCl, HFo (9:8:1.5:0.2:0.2)[22]

5) DCM, EtOAc, EtOH, HFo (3.5:2:4:0.5) [23]

6) EtOAc, MeOH, NH3 (8.5:1.5:0.5) [24]

Results are shown in table 3-6.

Table 3-6 RF values of anti-rheumatic drugs developed with solvent systems from literature

Method 1) 2) 3) 4) 5) 6) Dexamethasone 0.30 0 0.01 0.37 0.89 0.64Prednisone 0.30 0 0.05 0.31 0.86 0.59Prednisolone 0.30 0 0 0.25 0.86 0.57Propylphenazone 0.40 0.09 0.33 0.56 0.90 0.77ASA 0.30 0.12 0.02 0.49 0.90 degradedAcetaminophen 0.30 0 0.02 0.34 0.84 0.58

None of these methods could reliably separate NSAIDs and corticosteroids. Either

substances ran in the front, did not move at all or corticosteroids and Acetaminophen

could not be separated.

3.2.2. Method development

According to the CAMAG optimization scheme (see figure 1-2) a method for separa-

tion of NSAIDs and corticosteroids was developed. In a first step pure solvents from

seven selectivity groups were used. RF values are displayed in table 3-7.

44

Table 3-7 RF values of anti-rheumatic drugs developed with pure solvents

Solvent THF MeOH TBME Toluene EtOAc DCM CHCl3 Dexamethasone 1 0.91 0.61 0 0.81 0 0Prednisone 1 0.89 0.43 0 0.66 0 0Prednisolone 1 0.90 0.43 0 0.64 0 0Propylphenazone 1 0.86 0.88 0 0.88 0 0.16ASA 1 0.88 degraded 0 degraded 0 0Acetaminophen 1 0.92 0.5 0 0.69 0 0

In a second step solvents were modified with hexane or acetic acid. RF values are

shown in table 3-8.

Table 3-8 RF values of anti-rheumatic drugs developed with modified solvents

Solvent Hexane, THF (1:4)

Hexane, MeOH (1:4) Tol, HAc (5:1)

CHCl3, HAc (4:1)

DCM, HAc (4:1)

Dexamethasone 0.96 1 0.04 0.46 0.52Prednisone 0.91 1 0.04 0.52 0.53Prednisolone 0.89 1 0.03 0.29 0.27Propylphenazone 0.93 1 0.38 1 1ASA degraded 1 0.54 1 1Acetaminophen 0.77 1 0.06 0.30 0.40

In a third step binary solvent systems were tested. RF values of some mixtures are

viewable in table 3-9.

Table 3-9 RF values of anti-rheumatic drugs developed with binary solvent mixtures

Solvent 2-propanol, Tol (6:1)

EtOAc, CHCl3 (1:1)

TBME, DCM (9:1)

MeOH, Tol (2:8)

Heptan, THF (1:3)

Dexamethasone 1 0.26 0.49 0.25 0.50Prednisone 1 0.21 0.37 0.27 0.45Prednisolone 1 0.13 0.30 0.24 0.44Propylphenazone 1 0.80 0.88 0.39 0.57ASA 1 degraded degraded degraded 0.42Acetaminophen 1 0.27 0.51 0.23 0.33

In a last step complex solvent mixtures were tested. Results are shown in table 3-10.

Table 3-10 RF values of anti-rheumatic drugs developed with complex solvent mixtures

Solvent THF, heptane, HFo (5:5:0.5) Tol, 2-propanol, HFo (15:5:0.5) Tol, 2-propanole, NH3 (15:5:0.1)

Dexamethasone 0.25 0.41 0.54Prednisone 0.21 0.41 0.55Prednisolone 0.21 0.35 0.46Propylphenazone 0.41 0.54 0.72ASA 0.46 0.53 0.03Acetaminophen 0.20 0.29 0.42

45

ASA is an acid with a pKa value of 3.5. Thus it can be partially ionized. For the chro-

matography of ASA, it is crucial to add an acid to the solvent system.

As none of the mixtures produced satisfactory results, a solvent system for NSAIDs

was developed based on the solvent systems tested before. RF values are apparent

in table 3-11.

For corticosteroids a Method C (according to [17]) was tested. RF values are shown

in table 3-12.

.

Table 3-11 RF values of different NSAIDs developed with complex solvent mixtures

Solvent THF, heptane, HFo (5:5:1)

THF, heptane, HFo (5:5:1)

TBME, EtOAc, HFo (4.5:5:0.5)

DCM, EtOAc, HFo (5:4.5:0.5)

Propylphenazone 0.44 0.46 0.69 0.51ASA degraded 0.51 0.76 0.55Acetaminophen 0.3 0.3 0.36 0.39Caffeine 0.17 0.15 0.15 0.23Diclofenac 0.53 0.55 0.85 0.71Mefenamic acid 0.57 0.58 0.86 0.76Ibuprofen degraded 0.58 0.83 0.73

Solvent

Tol, EtOAc, HAc (60:10:1)

Heptane, 2-propanol, HFo (15:5:0.5)

Heptane, 2-propanol, HAc (15:5:1)

Propylphenazone 0.11 0.38 0.37 ASA 0.13 0.43 0.42 Acetaminophen 0 0.17 0.17 Caffeine 0 0.09 0.06 Diclofenac 0.22 0.54 0.54 Mefenamic acid 0.40 0.56 0.57 Ibuprofen degraded 0.55 0.56

Table 3-12 RF values of corticosteroids

Solvent DCM, Acetone (3:1)

Dexamethasone 0.21Prednisone 0.25Prednisolone 0.13

46


For analysis of synthetic substances in herbal pain killers, different samples were

examined.

Artificial samples were created to check the ability of an herbal matrix to cover syn-

thetic adulterants.

Four traditional Korean pain killers were purchased in Korea. One of them claimed to

combine western and eastern medicine and therefore contained Caffeine, Aceta-

minophen and Ethenzamide.

One pain killer decoction was purchased via the internet.

The samples were analyzed with Method B. Developing solvent was heptan, 2-

propanol, acetic acid (15:5:1). Plates were evaluated visually and with UV spectra

comparison.

Results are resumed in figures 3-10 and 3-11.

Figure 3-10 NSAIDs: Standards, artificial and real samples under UV at 254 nm

1: R2073-01-10% (Propylphenazone), 2: R2074-01 (ASA), 3: R2075-01-10% (Acetaminophen), 4: R2084-01-10% (Caffeine) 5: R2085-01-33% (Diclofenac), 6: R2086-01-50% (Mefencamic acid), 7: R2087-01 (Ibuprofen), 8: S2101-02 (Artificial mix 1), 9: S2102-02 (Artificial mix 2), 10: S2103-02 (Arti-ficial mix 3), 11: S2110-01 (Panosin), 12: S2111-01 (Maroicin), 13: S2112-01 (Yorosin), 14: S2113-01-10% (Iszpen), 15: S2116-50% (Comdrugcide)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

47

Figure 3-11 NSAIDs: Standards, artificial and real samples under UV at 254 nm

(Caption see above fig. 3-10)

Tracks 1 to 7 show the seven reference substances, tracks 8 to 15 eight different

samples.

The artificial sample mix 1 on track 8 has spots on the same height as standards Caf-

feine, Acetaminophen and Acetylsalicylic acid (ASA) respectively. Spectra compari-

son confirmed the presence of those ingredients. The fourth spot on the top may be a

degradation product of ASA.

The artificial sample mix 2 on track 9 shows a band on the height of Ibuprofen. Under

UV at 366 nm this spot shows a light blue fluorescence that the reference substance

on track 7 doesn’t have. But the UV spectra are identical. The fluorescence in the

sample might be caused by the higher concentration.

The two spots on track 10 correspond to the reference substances Propylphenazone

and Mefenamic acid respectively.

On track 14 the first band corresponds to the reference caffeine, the second to the

substance Acetaminophen. The UV spectrum of the third band was compared to

data in literature [25] and identified as Ethenzamide.

Under UV at 254 nm samples on track 11, 12, 13 and 15 show only weak bands. At

366 nm several blue fluorescent spots appear. UV spectra of all of them were com-

pared to the reference spectra. The spectra of the spot at RF value 0.46 on track 15

resembled to the spectra of Ibuprofen and ASA (see figure 3-12)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

48

Figure 3-12 Spectra comparison: References Ibuprofen and ASA and sample S2116-50%

For identification of corticosteroids method C was used. Results are displayed in fig-

ure 3-13.

Figure 3-13 Corticosteroids: Standards, artificial and real samples under UV at 254 nm

1: R2064-01-10% (Dexamethasone), 2: R2065-01-10% (Prednisone), 3: R2066-01-10% (Predniso-lone), 4: S2102-02 (Artificial mix 2), 5: S2110-01 (Panosin), 6: S2111-01 (Maroicin), 7: S2112-01 (Yo-rosin), 7a: S2112-01-Prep (isolated substance from Yorosin), 8: S2113-01-10% (Iszpen), 9: S2116-SPE (Comdrugcide), 9a: S2116-SPE-Prep (isolated substance from Comdrugcide) Tracks 1 to 3 show the three reference substances, tracks 4 to 9 six samples.

In the artificial sample on track 4, the two spots on the height of Dexamethasone and

Prednisone could also be identified as such with spectra comparison, whereas on

track 8 spectra comparison excluded the presence of a corticosteroid.

1 2 3 4 5 6 7 7a 8 9 9a

49

3.2.4. Results from NMR-, MS- and IR-analysis

The substance in sample S2116 Comdrugcide pain decoction, which had a UV spec-

trum similar to the reference substance, was isolated with Method F (see chapter

2.2.3.). An NMR-, MS- and IR-spectrum was taken.

Results are displayed in Annex 6.3. to 6.5.

The structure could not completely be identified, as it was not possible to isolate the

required amount in an adequate purity for C-NMR analysis. However it seems to be

clear that a COOH-group is present (cp. NMR-spectrum). The peak at wavenumber

1559 in the IR-spectrum indicates the existence of a nitro-group. The measured mo-

lecular mass of 337.1 seems to be rather high for the suspected NSAIDs, which nor-

mally range between 200 and 300 g/mol. Tenoxicam, an anti-rheumatic drug, has a

molecular weight of 337.4. After comparison with chemo-physical data in literature,

this substance could be excluded. Newer NSAIDs, so known COX2-Inhibitors, tend

to have a higher molecular weight (from 314 g/mol). But it is very unlikely that the

isolated compound belongs to this group, as these drugs have only been on the mar-

ket for a short time.

50

3.2.5. Discussion

For separation of NSAIDs solvent mixture heptane, 2-propanol, HAc (15:5:1) showed

best results. Visualization was made under UV at 254 nm and 366 nm. Under UV at

366 nm Mefenamic acid shows a bright blue fluorescence and can therefore be dis-

tinguished from Ibuprofen and Diclofenac which have a similar RF value.

It was found that ASA is instable in solution. Solutions should not be stored longer

than a week.

For separation of corticosteroids the method from literature showed satisfactory re-

sults. Detection was performed under UV at 254 nm.

The synthetic active ingredients in the artificial mixes (samples S2101 to S2103)

could be detected visually and with spectra comparison.

In sample S2101 ASA had been partially degraded and a second spot was visible

just above the original spot.

In the Korean samples claimed to be purely herbal (samples S2110 to S2112), no

synthetic adulterant could be found.

In the Korean sample declared to contain Ethenzamide, Acetaminophen and Caf-

feine (sample S2113) the active ingredients could be detected. For the lack of the

reference substance Ethenzamide, identification was achieved by spectra compari-

son with data from literature [25].

The sample S2116 purchased via internet showed a band with a UV spectrum re-

sembling to that of Ibuprofen to a certain extent. Despite further analytical investiga-

tions, the structure could not be determined.

For separation of corticosteroids solvent system from literature showed good results.

In the artificial mix 2, the added active ingredients could be detected visually and with

spectra comparison. In sample S2113 on track 8 and S2116 on track 9, the spots at

height of Dexamethasone and Prednisone respectively showed a different UV spec-

trum. None of the real sample contained corticosteroids.

51

3.3. Method development for anti-diabetic drugs

3.3.1. Existing method

An existing method for separation of different oral anti-diabetic drugs described in

literature was tested [26]. Developing solvent was phosphate buffer pH6 R2, 2-

propanol (1:1), as stationary phase an RP18 glass plate was used.

The separation of Metformin and Glibenclamide was successful, but Metformin

showed tailing.

3.3.2. Method optimization

3.3.2.1. Developing solvent

To decrease tailing of Metformin, phosphate buffer was substituted for water and

HAc. Because of the increased proportion of water, RP18W glass plates were used

as stationary phase. The RF values of the two solvent systems are compared in table

3-13.

Table 3-13 RF values of anti-diabetic substances and samples developed with different solvent systems

1: R2062-02-02-10% (Metformin), 2: R2063-02-02 (Glibenclamide), 3: S2107-01 (Mix 4), 4: S2108-01 (Mix 5), 5: S2109-01 (D-Abin), 6: S2117-01-02 (Xiaoke), 6a: S2117-02 (Xiaoke), 7: S2118-01 (Ye Tianshi)

Solvent

Phosphate buffer pH6 R2, 2-propanol

(1:1)

Water, 2-propanol, HAc (20:10:5)

Glibenclamide 0.44 0.22Metformin 0.55 0.68

1 2 3 4 5 6 7 1 2 3 4 5 6 6a 7

52

Acetic acid in the developing solvent sharpens bands and increases separation of

Metformin and Glibenclamide. Further tests were performed with water, 2-propanol,

HAc (20:10:5) as solvent system and RP18W glass plates as stationary phase (cp.

method D, chapter 2.2.3.)

3.3.2.2. Derivatization reagent

To stain oral anti-diabetic substances, different derivatization reagents were tested.

Results are listed below in table 3-14.

Table 3-14 Derivatization reagents

1: R2062-02-02-10% (Metformin), 2: R2063-02-02 (Glibenclamide), 3: S2107-01 (Mix 4), 4: S2108-01

(Mix 5), 5: S2109-01 (D-Abin), 6: S2117-01-02 (Xiaoke), 6a: S2117-02 (Xiaoke), 7: S2118-01 (Ye

Tianshi)

Method I (p-Chloranil) Method II (Anisaldehyd) Method III (H2SO4)

After derivatization with p-Chloranil reagent Sulfonylurea anti-diabetic agents should

be visible under white light. Under UV at 366 nm the sulfuric acid in the reagent

causes a blue fluorescence. The light brown spot on track 2 is not Glibenclamide but

might come from the tablet matrix.

Anisaldehyd reagent is a suitable reagent for various substances. Under UV at 366

nm a blue fluorescence is visible.

After derivatization with sulfuric acid reagent Glibenclamide shows a bright blue fluo-

rescence under UV at 366 nm.

1 2 3 4 5 6 6a 7

Solvent: 2-propanol, water, HAc (10:20:5)

under UV at 366 nm

1 2 3 4 5 6 7

Solvent: 2-propanol, phosphate buffer (1:1)

under UV at 366 nm

1 2 3 4 5 6 7

Solvent: 2-propanol, phosphate buffer (1:1)

under UV at 254 nm

53


For analysis of synthetic substances in herbal medicine different samples were ex-

amined.

Artificial samples were created to check the ability of an herbal matrix to mask syn-

thetic adulterants.

One traditional Korean anti-diabetic drug was purchased in Korea.

Two anti-diabetic drugs were purchased via the internet. One of them claimed to con-

tain the prescription-only substance Glibenclamide.

Pictures of the analyzed samples are displayed on figures 3-14 and 3-15.

Figure 3-14 Anti-diabetic substances and samples under UV at 254 nm

Figure 3-15 Anti-diabetic substances and samples under UV at 366 nm after derivati-zation with H2SO4 reagent

1: R2062-02-02-10% (Metformin), 2: R2063-02-02 (Glibenclamide), 3: S2107-01 (Mix 4), 4: S2108-01 (Mix 5), 5: S2109-01 (D-Abin), 6: S2117-01-02 (Xiaoke), 7: S2118-01 (Ye Tianshi)

On track 1 and 2 the two standards were applied. Metformin on track 1 is only visible

under UV at 254 nm. Glibenclamide on track 2 is visible under UV at 254 nm and af-

ter derivatization under UV at 366 nm (blue fluorescent spot) at an RF value of 0.18.

On track 3 to 7 fife different samples were applied. The two artificial samples on track

3 and 4 show multiple spots. Mix 4 on track 3 has a weak band at the same RF value

as the reference substance Glibenclamide. After derivatization under UV at 366 nm

the band has a light blue fluorescent. Mix 5 on track 4 shows a spot on the same

height as the reference substance Metformin.

Synthetic ingredients in Mix 4 and 5 could also be affirmed with spectra comparison.

1 2 3 4 5 6 7 1 2 3 4 5 6 7

54

Samples on track 5 and 6 show a complex pattern after derivatization. The Korean D-

Abin on track 5 has a blue fluorescent zone at RF value 0.18 (see figure 3-15). Under

UV at 254 nm (before derivatization) no such spot is identifiable. Spectra comparison

confirmed that the spot has not been generated by Glibenclamid.

Under UV at 254 nm sample Xiaoke on track 6 has a pail spot at RF value 0.18. After

derivatization no blue fluorescence is seen. Spectra comparison could not discover

the active ingredient either.

Sample Ye Tianshi on track 7 shows only one spot at RF value 0.78, which is proba-

bly caused by the plant matrix.

As Glibenclamide could not be detected in sample S2117 despite the manufacturer’s

declaration, further investigations were made.

First the sample preparation was examined. Sample S2117-02 was prepared as de-

scribed by Ku, Y.R. et al. [9]. Glibenclamide could not be deteceted.

In a second step the sample was spiked with the active ingredient in the declared

dosage. Glibenclamide could be detected.

At last sample S2117-01-02 was oversprayed with the reference substance to check

whether the plant matrix could disguise the synthetic adulterant. The oversprayed

part was compared visually and with spectra comparison to the reference substance

and the “blank” sample. Results are displayed in figures 3-16 to 3-18.

Figure 3-16 Oversprayed sample Xiaoke under UV at 254 nm

1: R2063-02-02 (Glibenclamide), 2: S2117-01-02 (Xiaoke oversprayed with R2063-02-02) , 3: S2117-01-02

1 2 3

55

Figure 3-17 Spectra comparison of over-sprayed sample

Figure 3-18 Spectra comparison of over-sprayed sample with adjusted application volume

On figure 3-16 on track 1, only the reference substance Glibenclamide was applied.

On track 2, Glibenclamide was applied as on track one, but was afterwards over-

sprayed with a large amount of sample S2117. On track 3, only sample S2117 was

applied.

The UV spectrum of the reference substance is colored in purple, the one of the

sample is colored in orange. The blue chart is the hybrid spectrum of the over-

sprayed part. The spectrum is mainly similar to the reference substance. The influ-

ence of the sample spectrum is visible on the generally upwards shifted line in com-

parison to the reference spectrum, as well as on the bulge at wavelength 265 nm.

In a second test, application volume where adjusted, so that they correspond to the

declared content. On figure 3-18 is visible, that a high amount of the sample is able

to alter the spectrum of the oversprayed zone, so that the reference substance is

hardly recognizable.

56

3.3.4. Discussion

For the detection of synthetic anti-diabetic substances, 2-propanol, water, HAc

(10:20:5) showed best results. Glibenclamide is a high potency drug and therefore

taken only at low dosage. This makes detection difficult. To color Glibenclamide and

other Sulfonylurea anti-diabetic drugs, derivatization with H2SO4 in MeOH (1:9) is

suitable.

In the artificial samples the added active ingredients could be detected.

In the samples claimed to be purely herbal (S2109 and S2118), no synthetic adulter-

ant was found.

Sample Xiaoke (S2117) declared to contain Glibenclamide. Despite different analysis

this active ingredient could not be determined. Test with overspraying showed that

the sample matrix has an influence on the spectrum of the active ingredient and

could be able to mask the substance.

In a next step this sample should have been analyzed with LC/MS, to check whether

other chromatographic methods yield the same results.

57

3.4. Final Discussion

Three methods for analysis of synthetic adulterants in herbal medicines were devel-

oped. Evaluation was always achieved visually and with UV spectra comparison.

For anti-impotency drugs the solvent system tert-Butyl methyl ether, methanol, am-

monia (20:2:1) showed best results. Stationary phase was a common silica gel plate.

Six samples were analyzed. Three of them contained illegally added Sildenafil and

Homosildenafil respectively.

Heptane, 2-propanol, acetic acid (15:5:1) was found to be appropriate for analytics of

anti-rheumatic drugs. For corticosteroids dichloromethane, acetone (3:1) led to ade-

quate separation. Stationary phases were in both cases silica gel plates. Eight sam-

ples were analyzed. The declared synthetic ingredients in the three artificial samples

and in one Korean pain killer could be detected. Three samples did not contain any

synthetic adulterants. One sample probably contained an adulterant, but its identity

could not definitely be elucidated.

For anti-diabetic agents the solvent system 2-propanol, water, acetic acid (10:20:5)

was suitable. As stationary phase RP18W plates were used. Fife samples were ex-

amined. The active ingredients in the two artificial samples were recovered. Other

two samples did not contain any synthetic adulterant. One sample declared to con-

tain an active ingredient that could not be detected by HPTLC. In this work it could

not be determined whether the substance was not in the sample or it could not be

discovered.

In a future project the quantitative evaluation should be re-investigated with special

respect to stability of the components. Preparative isolation of potentially synthetic

substances from the investigated herbal drugs in larger quantities should be at-

tempted. Elucidation of structures would then be possible

59

4. List of Figures

Figure 1-1 Method development schema................................................................... 13

Figure 1-2 CAMAG optimization scheme................................................................... 13

Figure 3-1 Anti-impotency drugs, standards and samples......................................... 33

Figure 3-2 Anti-impotency drugs, standards and samples; optimized solvent........... 33

Figure 3-3 Standard solutions and samples from Korean custom............................. 34

Figure 3-4 UV spectrum of standard Sildenafil and samples from Korean custom .. 34

Figure 3-5 UV spectrum of standard Homosildenafil and sample S1922 ................. 34

Figure 3-6 Standard solutions and samples purchased in Hong Kong...................... 35

Figure 3-7 UV spectrum of Standard Sildenafil and analogues and sample S1925.. 35

Figure 3-8 UV spectrum of Standard Sildenafil and analogues and sample S1926.. 35

Figure 3-9 Calibration curve of Sildenafil and measurements of sample S1921....... 41

Figure 3-10 NSAIDs: Standards, artificial and real samples under UV at 254 nm .... 46

Figure 3-11 NSAIDs: Standards, artificial and real samples under UV at 254 nm .... 47

Figure 3-12 Spectra comparison: References Ibuprofen and ASA and sample………..

S2116-50%.............................................................................................. 48

Figure 3-13 Corticosteroids: Standards, artificial and real samples under UV at ……...

254 nm..................................................................................................... 48

Figure 3-14 Anti-diabetic substances and samples under UV at 254 nm.................. 53

Figure 3-15 Anti-diabetic substances and samples under UV at 366 nm after…………

derivatization with H2SO4 reagent ........................................................... 53

Figure 3-16 Oversprayed sample Xiaoke under UV at 254 nm ................................. 54

Figure 3-17 Spectra comparison of oversprayed sample .......................................... 55

Figure 3-18 Spectra comparison of oversprayed sample with adjusted application…...

volume ..................................................................................................... 55

60

5. Literature References 1. Koh, H.L., Woo, S.O., 2000. Chinese proprietary medicine in Singapore: regu latory control of toxic heavy metals and undeclared drugs. Drug Safety, 23(5): p. 351-62. 2. Yee, S.K., Chu, S.S., Xu, Y.M., 2005. Regulatory control of Chinese Proprie tary Medicines in Singapore. Health Policy, 71(2): p. 133-49. 3. Chan, K., 2003. Some aspects of toxic contaminants in herbal medicines.

Chemosphere, 52(9): p. 1361-71. 4. Ernst, E., 2002. Adulteration of Chinese herbal medicines with synthetic drugs:

a systematic review. Jounal of Internal Medicine, 252(2): p. 107-13. 5. De Smet, P.A., 2004. Health risks of herbal remedies: An update. Clinical Pharmacology & Therapeutics, 76(1): p. 1-17. 6. Gertner, E., Marshall, P.S., Filandrinos, D., Potek, A.S., Smith, T.M., 1995.

Complications resulting from the use of Chinese herbal medications containing undeclared prescription drugs. Arthritis and Rheumatism, 38(5): p. 614-7.

7. Huang, W.F., Wen, K.C., Hsiao, M.L., 1997. Adulteration by synthetic thera peutic substances of traditional Chinese medicines in Taiwan. Journal of Clini cal Pharmacology, 37(4): p. 344-50. 8. Ko, R.J., 1998. Adulterants in Asian patent medicines. New England Journal of Medicine, 339(12): p. 847. 9. Ku, Y.R., Chag, L.Y., Ho, L.K., Lin, J.H., 2003. Analysis of synthetic anti-

diabetic drugs in adulterated traditional Chinese medicines by high-performance capillary electrophoresis. Journal of Pharmaceutical and Bio-medical Analysis, 33(2): p. 329-34.

10. Schnädelbach, D., 1999. The counterfeiting of pharmaceuticals: containing

risks., in The Pharmacists at the crossroads of new health risks: An indispen-sable partner for their man agement. Strasbourg, France, Council of Europe.

11. Hong, M.-K., et al., 2005. Private communication, CAMAG. 12. Reich, E. and A. Blatter, 2004. Modern TLC: A key technique for identification

and quality control of botanicals and dietary supplements. Inside Laboratory Management, May/June p. 14-18.

13. Reich, E., 2003. Instrumental Thin-layer Chromatography, in Handbook of

Thin-layer Chromatography, Sherma, J., Fried, B. (Eds). Marcel Dekker, Inc., New York, p. 135-151.

61

14. Sherma, J., 2002. Chromatographic Methods of Analysis - Thin Layer Chro-matography in Encyclopedia of Pharmaceutical Technology, D. Encyclocedia, (Ed.). Marcel Dekker Inc., New York, p. 426-439.

15. Snyder, L.R., 1978. Classification of the solvent properties of common liquids.

Journal of Chromatographic Science, 16: p. 223-234. 16. Dammertz, W., Reich, E., 2001. Planar chromatography and densitometry, in

Planar chromatography, S. Nyiredy (Ed.). Springer Scientific Publisher, Buda-pest, Hungary, p. 234 - 246.

17. Pachaly, P., 1989. Steroidhormon-Derivate. Deutsche Apotheker Zeitung. 129:

p. 1538-1539. 18. Jork, H., Funk, W., Fischer, W., Wimmer, H., 1994. Thin-Layer Chromatogra-

phy: Reagents and Detection Methods. VHC Verlagsgesellschaft mbH. 19. European Pharmacopoeia. 4 ed. Swiss Edition, 2002. Deutscher Apotheker

Verlag, Stuttgart. 20. Sethi, P.D., Charegaonkar, D., 1999. Musculoskeletal Disorders, in Identifica-

tion of drugs in pharmaceutical formulations by thin-layer chromatography, CBS Publishers & Distributors, New Delhi, India. p. 83-132.

21. CAMAG, 2003. Quantitation of acetylsalicylic acid, salicylic acid and salicyla-

mide by HPTLC. 22. Franeta, J.T., Agbaba, D.D., Eric, S.M., Pavkov, S.P., Vladimirov, S.D., Alek-

sic, M.B., 2001. Quantitative analysis of analgoantipyretics in dosage form us-ing planar chromatography. Journal of Pharmaceutical and Biomedical Analy-sis, 24(5-6): p. 1169-1173.

23. El Sadek, M., El Shanawany, A., Aboul Khier, A., Ruckner, G., 1990. Determi- nation of the components of analgesic mixtures using high-performance thin- layer chromatography. Analyst, 115(9): p. 1181-1184. 24. Bebawy, L.I., El-Kousy, N.M., 1999. Simultaneous determination of some mul-

ticomponent dosage forms by quantitative thin layer chromatography densi-tometric method. Journal of Pharmaceutical and Biomedical Analysis, 20(4): p. 663-670.

25. http://chrom.tutms.tut.ac.jp/JINNO/DRUGDATA/14ethenzamide.html. Last vis-

ited on 21.09.2005. 26. Gumieniczek, A., Hopalka, H., Berecka, A., Kowalczuk, D., 2003. Normal-

and reversed-phase thin-layer chromatography of seven oral antidiabetic agents. Journal of Planar Chromatography, 16: p. 271-275.

62

6. Annex

6.1. List of Abbreviations

ACN Acetonitrile

ADC Automatic Development Chamber

AMD Automated multiple development

ASA Acetylsalicylic acid

CHCl3 Chloroform

CH2Cl2 Dichloromethane

CAM Complementary and alternative Medicine

DCM Dichloromethane

DMF Dimethylformamide

EtOAc Ethyl acetate

EtOH Ethanol GC Gas chromatography

GMP Good Manufacturing Practice

HAc Acetic acid

HCl Hydrochloric acid

HFo Formic acid

H2SO4 Sulfuric acid

HPLC High Performance Liquid Chromatography

HPTLC High Performance Thin Layer Chromatography

IR Infrared

MEK Methyl ethyl ketone

MeOD Deuterized methanol

MeOH Methanol

MS Mass Spectrometry

NaOH Sodium hydroxide

NH3 Ammonia 28%

NMR Nuclear Magnetic Resonance

NSAID Non-steroidal anti-inflammatory drug

p.a. pro analysi

63

RF Retention Factor

RP Reversed Phase

SPE Solid Phase Extraction

TBME tert-Butyl methyl ether

TCM Traditional Chinese Medicine

THF Tetrahydrofuran

TLC Thin Layer Chromatography

Tol Toluene

TTC Twin Trough Chamber

UV Ultraviolet

WRT White light Reflection and Transmission

64

6.2. LC/MS spectra of anti-impotency drugs

Standard R1932 Sildenafil

65

Sample S1921

66

Sample S1925-02-SPE

12 3

45

6

67

68

69

70

Sample S1926-01-SPE-Prep

1

2

71

72

6.3. NMR spectrum of anti-rheumatic drug

73

6.4. MS spectrum of anti-rheumatic drug

74

75

76

77

78

79

80

6.5. IR spectrum of anti-rheumatic drug

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