1

CHAPTER-1

General Introduction

2

The search for remedies is as old as humanity itself. There is evidence that the first

advanced civilizations were already using drugs of plant, mineral and animal origin for

medicinal purposes. Systematic descriptions of remedies have been handed down to us

from Greek antiquity and from the Roman Empire. This knowledge was adopted by

Arabian scholars and was developed further. It served for a long time as an important

basis of medicine. The gradual change from the ancient models started in 16th

century. A

typical representative of the new direction was Paracelsus, lived in Basel, who, in 1537,

coined the famous phrase: “only dose makes the poison”. The emergence of organic

chemistry at the start of 19th

century brought progress. In 1906, P. Ehrlich discovered a

chemical compound named atoxyl which had been shown to be able to treat sleeping

sickness. Ehrlich received the Nobel Prize for medicine in 1908. The period from

Paracelsus to Ehrlich has been described as that leading from Quintessence to the

Chemical and has been reviewed by Barber [1]. This transition from Quintessence to

Chemical stimulated a considerable amount of interest in the analysis as well as

determination of purity of natural products.

Pharmaceutical industry is growing day by day with the aim to develop new drugs

extracted from natural products or synthetically produced drug substances, but one thing

always remains constant, that is, the product should be as pure as possible. Therefore,

purity has always been considered as an essential factor in ensuring drug quality. In

pharmaceutical industry, the quality of the manufactured drug and its formulations must

be carefully controlled. Slight changes in composition or in the purity of drugs itself can

affect the therapeutic values. Therefore, it is necessary to establish the properties and

therapeutic value of a drug before it is approved and made available in the market.

Establishment of the permissible level of dosage of a drug requires the determination of

its composition, toxicity and its metabolites at various stages.

3

The availability of sub-standard medicines to the general public possesses many

problems, both clinically and economically. It is widely believed that sub-standard drug

preparations are readily available in many developing countries [2-6]. This may be due to

poor manufacturing procedures, poor storage conditions or deliberate counterfeiting of

branded or generic products. Therefore, it is important to recognize that the drugs may

contain impurities.

IMPURITY PROFILING

There is an ever increasing interest in impurities present in active pharmaceutical

ingredients (API’s). Recently, not only purity profile but also impurity profile has

become essential as per various regulatory requirements. In the pharmaceutical world, an

impurity is considered as any other organic material, besides the drug substance, or

ingredients, arise out of synthesis or unwanted chemicals that remains with API’s. The

impurity may be developed either during formulation, or upon aging of both API’s and

formulated API’s in medicines. The presence of these unwanted chemicals, even in small

amount, may influence the efficacy and safety of the pharmaceutical products. Impurity

profiling (i.e., the identity as well as the quantity of impurity in the pharmaceuticals), is

now gaining critical attention from regulatory authorities.

The different Pharmacopoeias, such as the British Pharmacopoeia (BP), United

States Pharmacopeia (USP), and Indian Pharmacopoeia (IP) are slowly incorporating

limits to allowable levels of impurities present in the API’s or formulations. The

International Conference on Harmonization of Technical Requirements for Registration

of Pharmaceuticals for Human Use (ICH) has also published guidelines for validation of

methods for analysing impurities in new drug substances, products, residual solvents and

microbiological impurities [7-10].

4

Qualification of impurities is the process of acquiring and evaluating data that

establishes biological safety of an individual impurity; thus, revealing the need and scope

of impurity profiling of drugs in pharmaceutical research (Fig. 1.1).

Classification of impurities

Impurities in the drug substance produced by chemical synthesis can broadly be

classified into following three categories:

a) Organic impurities: It may arise during the manufacturing process and/or storage of

the drug substance. They may be identified or unidentified, volatile or non-volatile, and

may include:

starting materials or intermediates

by-products

degradation products

reagents, ligands, and catalysts (less commonly found in APIs but in some cases

they may pose a problem as impurities)

b) Inorganic impurities: These impurities are generally known and identified and may

originate from the following sources:

the use of equipments,

reagents, ligands, and catalysts, that are used during production, may serve as

impurities,

the water used in the processes and the stainless steel reactors, where acidification

or acid hydrolysis takes place, may discharge heavy metals as impurities,

contain drying agents that are generally employed during production,

5

Fig. 1.1. Proposed chart for profiling drug impurity.

6

the use of filter aids (such as centrifuge bags or activated charcoal that are

routinely used in the bulk manufacturing plants).

c) Residual solvents: These are organic or inorganic volatile liquids used during the

manufacturing process or generated during the production.

Origin of Impurities from different sources

Crystallization-related impurities

Based on the realization that the nature of structure adopted by a given compound

upon crystallization could exert a profound effect on the solid-state properties of that

system, the pharmaceutical industry is required to take a strong interest in polymorphism

and solvatomorphism [11] as per the regulations laid down by the regulatory authorities.

Stereochemistry-related impurities

It is of paramount importance to look for stereochemistry related compounds; that

is, those compounds that have similar chemical structure but different spatial orientation,

these compounds can be considered as impurities in the API’s. The single enantiomeric

form of chiral drug is now considered as an improved chemical entity that may offer a

better pharmacological profile and an increased therapeutic index with a more favourable

adverse reaction profile. However, the pharmacokinetic profile of levofloxacin (S-

isomeric form) and ofloxacin (R- isomeric form) are comparable, suggesting the lack of

advantages of single isomer in this regard [12]. The prominent single isomer drugs, which

are being marketed, include levofloxacin (S-ofloxacin), levalbuterol (R-albuterol), and

esomeprazole (S- omeprazole).

7

Residual solvents

Some residual solvents that are known to cause toxicity should be avoided in the

production of bulk drugs. Depending on the possible risk to human health, residual

solvents are divided into three classes [13]. Class I includes solvents such as, benzene (2

ppm limit), carbon tetrachloride (4 ppm limit), 1,2- dichloroethane (5 ppm limit), 1,1-

dichloroethene (8 ppm limit) and 1,1,1- trichloroethane (1500 ppm limit). The solvents

belong to class II are methylene chloride (600 ppm limit), methanol (3000 ppm limit) ,

pyridine (200 ppm limit), toluene (890 ppm limit), N, N- dimethylformamide (880 ppm

limit) and acetonitrile (410 ppm limit); which should be avoided. Class III solvents, viz

acetic acid, ethanol, acetone have permitted daily exposure of 50 mg or less per day, as

per the ICH guidelines.

Synthetic intermediates and by-products

Impurities in pharmaceutical compounds or a new chemical entity can originate

during the synthetic process from raw materials, intermediates and/or by-products. For

example, methamphetamine hydrochloride is one of the most widely used illicit drugs in

Philippines. The trace impurities in seized methamphetamine samples were identified

using gas chromatography-mass spectrometry and their quantification was made using

gas chromatography with flame ionization detector [14].

Formulation-related impurities

Many impurities in a drug product can originate from excipients used to formulate

a drug substance. In addition, a drug substance is subjected to a variety of conditions in

the process of formulation that can cause its degradation or have other undesirable

reactions. If the source is from an excipient, variability from lot to lot may make a

marginal product, unacceptable for reliability. Solutions and suspensions are inherently

prone to degradation due to hydrolysis or solvolysis [15]. In general, liquid dosage forms

8

are susceptible to both degradation and microbiological contamination. In this regard,

water content, pH of the solution/suspension, compatibility of anions and cations, mutual

interactions of ingredients, and the primary container are critical factors.

Impurities arising during storage

A number of impurities can originate during storage or shipment of drug products.

It is essential to carry out stability studies to predict, evaluate, and ensure drug product

safety [11].

Method related impurity

A known impurity, 1-(2, 6-dichlorophenyl) indolin-2-one is formed in the

production of a parenteral dosage form of diclofenac sodium, if it is terminally sterilized

by autoclave [16]. The conditions of the autoclave method (i.e., 123 ± 2 oC) enforce the

intramolecular cyclic reaction of diclofenac sodium forming an indolinone derivative and

sodium hydroxide. The formation of this impurity has been found to depend on initial pH

of the formulation.

Mutual interaction amongst ingredients

Most vitamins are very labile and on ageing they create a problem of instability in

different dosage forms, especially in liquid dosage forms. Degradation of vitamins does

not give toxic impurities; however, potency of active ingredients drops below

Pharmacopoeial specifications. Because of mutual interaction, the presence of

nicotinamide in a formulation containing four vitamins (nicotinamide, pyridoxine,

riboflavin, and thiamine) can cause the degradation of thiamine to a sub-standard level

within a one year shelf life of vitamin B complex injections [17].

Functional group-related typical degradation

Ester hydrolysis can be explained with a few drugs viz aspirin, benzocaine,

cefotaxime, ethyl paraben [17] and cefpodoxime proxetil [18]. Hydrolysis is the common

9

phenomenon for ester type of drugs, especially in liquid dosage forms viz

benzylpenicillin, oxazepam and lincomycin. Oxidative degradation of drugs like

hydrocortisone, methotrexate, hydroxyl group directly bonded to an aromatic ring (viz

phenol derivatives such as catecholamines and morphine), conjugated dienes (viz vitamin

A and unsaturated free fatty acids), heterocyclic aromatic rings, nitroso and nitrite

derivatives, and aldehydes (especially flavorings) are all susceptible to oxidative

degradation. In mazipredone, the hydrolytic and oxidative degredation pathways in 0.1

mol L-1

hydrochloric acid and sodium hydroxide at 800 °C were studied [19]. Photolytic

cleavage includes example of pharmaceutical products that are exposed to light while

being manufactured as solid or solution, packaged, or when being stored in pharmacy

shops or hospitals for use by consumers. Most compounds will degrade as solutions when

exposed to high-energy UV exposures. Fluoroquinolone antibiotics are also found to be

susceptible to photolytic cleavage [20]. In ciprofloxacin eye drop preparation (0.3 %),

sunlight induces photocleavage reaction producing ethylenediamine analog of

ciprofloxacin [21]. As seen earlier, impurities in drug products can come from the drug or

from excipients or can be brought into the system through an inprocess step by contact

with the packaging material. These impurities are needed to be analyzed by using

different analytical methods.

ANALYTICAL TECHNIQUES

Analysis of drugs in biological samples and pharmaceutical products is becoming

more important owing to the need to understand the therapeutic and toxic effects of drugs

and hence efforts are being made to develop more selective and effective drugs [22-25].

Because of the vast number of drugs introduced every year, more and more methods for

drugs and their metabolites determination are being developed either for routine or

research use. The assay procedures in official monographs have been described to

10

characterize the quality of bulk drug materials by setting limits of their active ingredient

content. The assay methods included in official monographs are titrimetry, spectrometry,

chromatography, capillary electrophoresis and electroanalytical methods. Based on the

European [26] and US Pharmacopoeias [27], the proportions of various analytical

techniques used in drug analysis are summarized in Table 1.1.

Titrimetric methods

Titrimetric methods, owing to its inherent simplicity, have been incorporated in

pharmacopoeias for determination of pharmaceuticals in drug formulations. Advantages

of these methods are saving time and labour, high precision and the fact that there is no

need of using reference standards. As can be seen from Table1.1 that 69.5 % and 40.5 %

of the total assay methods are devoted to titrimetric methods in European and US

Pharmacopoeias, respectively. Recently titrimetric method has been used for the assay of

quetiapine fumarate and tramadol hydrochloride in pharmaceutical formulations [28, 29].

High Performance Liquid Chromatography (HPLC)

As a vital part of drug development, pharmaceutical research and development has the

task of developing optimal stable and bioavailable formulations that allow evaluation of

new chemical entities in preclinical and clinical trials. HPLC has been the most powerful

and versatile tool for the detection and quantitation of chemical components in the

complex matrices frequently encountered in pharmaceutical analysis.

11

TABLE 1.1: Proportion of various analytical methods prescribed for the assay of

bulk drug materials in Ph. Eur. 4 [26] and USP XXVII [27].

Method Ph. Eur. 4

(%)

USP 27

(%)

HPLC

GC

Titration

Acid-base

Aqueous mixtures

Indicator

Potentiometric

Non-aqueous

Indicator

Potentiometric

Redox (Iodometry, Nitritometry, etc.)

Other (complexometry, argentometry, etc.)

UV-vis spectrophotometry

Microbiological assay (antibiotics)

Other (IR, NMR, polarimetry, fluorimetry, atomic

absorption spectroscopy, polarography, etc).

15.5 %

2 %

69.5 %

57.5 %

21 %

6.5 %

14.5 %

36.5 %

9.5 %

27 %

6.5 %

5.5 %

9.5 %

3 %

0.5 %

44 %

2.5 %

40.5 %

29.5 %

5.5 %

4.5 %

1 %

24 %

14 %

10 %

5.5 %

5.5 %

8.5 %

2.5 %

2 %

12

In general, there are two types of HPLC procedures in pharmaceutical analysis: the

method for the quantitation of the major component (drug substance) and the method for

the detection and/or quantitation of drug substance related impurities and degradants. As

can be seen in Table 1.1, this has become the predominant method in USP XXVII [27]

and-although to a lesser extent-it is one of the most widely used methods also in Ph. Eur.

4 [26]. The specificity of this method is excellent and at the same time sufficient

precision is also attainable. Fig. 1.2 shows the pie diagram indicating the percent usage of

various chromatographic techniques from which it becomes very clear that HPLC has

been the main technique used for analysis of impurities in drugs. Fig. 1.3 shows the

percent usage of various detectors used for HPLC analysis of drugs. The choice of proper

detection mode is crucial to ensure that all the components are detected. With UV

detection, this problem could be overcome by using a multiple wavelength scanning

programme which is capable of monitoring several wavelengths simultaneously. It

provides assurance that all the UV absorbing components are detected, if present in

sufficient quantity. Photodiode-array detectors are useful in determining the purity of

enantiomeric drugs by HPLC. Most workers used the reversed-phase mode with UV

absorbance detection whenever appropriate, because this provided the best available

reliability, repeatability and sensitivity.

Several drugs such as gentamicin sulphate [30], duloxetine hydrochloride [31],

mebeverine, mesalazine, sulphasalazine, aspirin [32] and tolvaptan [33] have been

assayed in pharmaceutical formulations and in biological fluids [34] using HPLC. Thus,

HPLC provides a major service in answering many questions posed by pharmaceutical

industry. However, the limitations of HPLC include the cost of columns, solvents and a

lack of long term reproducibility due to the proprietary nature of column packing. Liquid

chromatography combined with mass spectrometry (LC-MS) is considered as one of the

most important techniques of the last decade of 20th

century [35, 36]. Recently HPLC-

MS has been used for assay of drugs [37-40].

13

Fig. 1.2. Pattern of use of different chromatographic techniques employed for

analysis of impurities in drugs.

14

Fig. 1.3. Usage of different detectors for HPLC analysis of drugs.

15

Gas chromatography

Gas chromatography is a dynamic method for separation and detection of volatile

organic compounds. The advent of high- molecular weight products such as polypeptides,

or thermally unstable antibiotics limits the scope of this technique. Its principal limitation

rests in the relative non-volatility of the drug substances. Therefore, derivatization is

mandatory, but the techniques for producing volatile derivatives of drugs are legion. Due

to insufficient volatility and thermal stability of the majority of drug materials, gas

chromatography can also be used for their assay in a limited number of cases only, as

reflected by the figures in Table 1.1. Recently, gas chromatography has been used for

assay of drugs such as levetiracetam, [41], divalproex sodium [42] and residual benzene

in oral liquid pharmaceutical products [43].

Thin layer chromatography

Thin-layer chromatography (TLC) is a routine analytical technique, which finds

wide applications in the field of pharmaceutical analysis. TLC has distinct advantages

such as simplicity (require less sophisticated apparatus), minimal sample clean-up, wide

choice of mobile phases, flexibility in sample distinction, high sample loading capacity

and low cost. TLC is a powerful tool for screening unknown materials in bulk drugs

[44]. It provides a relatively high degree of assurance that all possible components of the

drug are separated. TLC has been exploited for quantitative analysis using spot elution

followed by spectrophotometric measurement. It has been utilized for impurity profiling

in pharmaceuticals [45] and for the determination of drugs such as amphetamine [46],

dutasteride [47], bromhexine hydrochloride, etofylline, salbutamol sulphate [48] and

azaphenothiazines [49].

16

High performance thin layer chromatography

High performance thin layer chromatography (HPTLC) is a fast separation

technique and flexible enough to analyze different kind of samples. This technique is

more advantageous in many ways as it is simple to handle and requires short analysis

time to analyze the complex or the crude sample clean up. HPTLC evaluates the entire

chromatogram with a wide variety of techniques and parameters without time constrains.

Moreover, there is simultaneous but independent development of multiple sample and

standards on each plate, leading to an increased reliability of results. HPTLC procedures

have been used for the simultaneous quantitation of famotidine and domeperidone [50]

lamivudine, stavudine, nevirapine [51] in bulk and dosage forms.

Capillary electrophoresis

Capillary electrophoresis (CE) is a relatively new analytical technique based on

the separation of charged analytes through a small capillary under the influence of an

electric field. In this technique solutes are seen as peaks as they pass through the detector

and the area of each peak is proportional to their concentration, which allows quantitative

determinations. CE separations are generally more efficient, can be performed on a faster

time scale, require only nanoliter injection volumes, and in most cases, take place under

aqueous conditions. These four characteristics of CE have proven to be advantageous for

many pharmaceutical applications. Several reports have appeared on the application of

this technique in the routine drug analysis [52-54].

Flow injection analysis (FIA)

Automation is a key demand in modern Analytical Chemistry. Process and quality

control require fast and reliable results in all areas of human activity. FIA technique [55]

became a versatile instrumental tool that contributed substantially to the development of

automation in pharmaceutical analysis. This can be well documented by a number of

17

reviews on the use of FIA in drug analysis [56-60]. Three key attributes of FIA ensured

its rapid development and wide acceptance:

(i) fundamental principles are easy to understand and implement, (ii) the instrumentation

can be readily assembled from simple, inexpensive and accessible components, and (iii) it

provides a simple means of automating many manual chemical analytical procedures.

Practically, FIA can be coupled with all methods of detection that are used in

contemporary chemical analysis. Following the general application of computers in

routine laboratory a second generation of flow analysis was proposed by Ruzicka and

Marshall in 1990 designated sequential injection analysis [61]. As with the FIA, this is a

non segmented continuous flow technique based on the same principle of controlled

dispersion and reproducible manipulation of the FIA concept, but whose mode of

functioning is based on the concept of programmable flow. The FIA technique has led to

continuously increasing interest in pharmaceutical analysis and some applications using

different detectors are summarized in Table 1.2.

UV-Visible spectrophotometry

UV-Visible spectrophotometry can be regarded as one of the suitable and

economical methods of drug analysis, although its importance has not decreased to any

extent during modern times. Moreover-mainly as a result of its coupling with other

methods-the field of application of spectrophotometry continues to increase. Methods

based on the natural ultraviolet-visible absorption of analyte are applied for the

determination of active substances in bulk drugs. This approach has certain limitations

from the points of the applicability, sensitivity, selectivity and effectiveness of the

measurement.

18

TABLE 1.2: Analysis of pharmaceuticals by FIA techniques.

Drug Detector Matrix References

Ambroxol Amperometric dosage form [55]

Ciprofloxacin Spectrophotometric dosage form [60]

Distigmine Potentiometric dosage form,

human urine

[62]

Fluoxetine Square-wave

adsorptive-sripping

voltammetric

dosage form [58]

Lansoprazole UV dosage form [56]

Metoclopramide Fast stripping

continous cyclic

voltammetric

dosage form [59]

Paracetamol Spectrophotometric dosage form [57]

Thiamine Fluorimetric dosage form [63]

19

Here one of the most important methods to extend the possibilities of spectrophotometric

analysis is presented, namely the combination of the spectrophotometric measurement

with a preliminary chemical reaction where the reaction product is the subject of

absorbance measurement. The chemical reaction is selected in such a way that the

absorption spectrum of the reaction product should be shifted toward the longer

wavelengths with occasional increase of its intensity as compared with that of parent

compound. It is apparent from Table 1.1 that the UV-Visible spectrophotometric

methods of analysis contribute to a larger extent in Ph. Eu 4 (9.5 %) and USP (8.5 %).

Several approaches using spectrophotometry for determination of active pharmaceutical

ingredients in bulk drug and formulations have been reported and details of these

methods are recorded in Table 1.3

Derivative spectrophotometry

In derivative spectrophotometry, spectra are obtained by plotting the first or

higher order derivative of absorbance or transmittance with respect to wavelength as a

function of wavelength. Often these plots reveal spectral detail that is lost in an ordinary

spectrum.

Moreover, it can be of use in quantitative analysis to measure the concentration of an

analyte whose peak is obscured by larger overlapping peak due to something else in the

sample. If two substances X and Y absorb in the same spectral region, then the

absorbance A = Ax + Ay. Its differentiation with respect to λ yields:

dA ⁄ dλ = bCx(dЄ/dλ)x

Thus the derivative is directly proportional to concentration. In a similar way, at λmax the

second derivative is also proportional to concentration. Excellent reviews of the theory

and applications of derivative spectrophotometry have been published by O’Haver [113]

and Fell [114]. Table 1.4 illustrates the analytical characteristics of derivative methods

for determination of pharmaceutical compounds [115-120].

20

TABLE 1.3: Quantitative analysis of drugs in pharmaceutical formulations by UV-

visible Spectrophotometric procedure.

Name of drug Reagents used λmax (nm) References

Acetaminophen m-Cresol 640 [64]

Amiodarone HCl p-Chloranilic acid

2,3-Dichloro 5,6-

dicyano1,4- benzoquinone

535

575

[65]

[65]

Amlodipine

besylate

p-Chloranilic acid 540 [66]

Ninhydrin in DMF

medium

595 [67]

2,3-Dichloro 5,6-

dicyano1,4- benzoquinone

580 [68]

Ascorbic acid 530 [68]

Amoxycillin &

ampicillin

KIO3 520 [69]

Ampicillin,

amoxycillin &

carbenicillin

Folin ciocalteau phenol 750,770

& 750

[70]

Ascorbic acid 1-Chloro-2,4-

dinitrobenzene

380 [71]

Diltiazem HCl Sodium metavanadate

Bromothymol blue

Bromophenol blue

Bromocresol green

750

415

415

415

[72]

[73]

[73]

[73]

Enalapril maleate KIO3 and KI

p-Chloranilic acid

2,3-Dichloro 5,6-

dicyano1,4-benzoquinone

352

510

565

[74]

[74]

[74]

Iodine 365 [74]

Esomeprazole

magnesium

5-Sulphosalicylic acid 365 [75]

Famotidine KMnO4 in alkaline

medium

610 [76]

Ninhydrin 590 [77]

Irbesartan Potassium iodate and 352 [78]

21

iodide in aqueous medium

Isoxuprine HCl Hydroxylamine

hydrochloride and

cerium(IV) nitrate

380 [79]

L-dopa NaOH 300 [80]

Labetalol HCl Sodium nitroprusside &

hydroxylamine

hydrochloride

695

[81]

4-Amino benzenesulfonic

acid, sodium nitrite and

sodium bicarbonate

395 [82]

Potassium permanganate 605 [83]

Levodopa Ce(IV) nitrate in H2SO4

medium

510 [84]

Lisinopril 7,7,8,8-

Tetracyanoquinodimethane

743 [85]

p-Chloranilic acid 525

[85]

Ninhydrin 595

[86]

Ascorbic acid 530

[86]

N-Bromosuccinimide

Chloranil

353

520

[87]

[87]

Losartan potassium KMnO4 in alkaline

medium

603 [88]

Menadione NaOH in the presence of

amine

450 [89]

Methyldopa Ce(IV) nitrate in H2SO4

medium

550 [90]

Metoprolol tartrate KMnO4 in alkaline

medium

610 [91]

Ninhydrin 595 [92]

Nalidixic acid Persulphate in alkaline

medium

320,390 [93]

Nicorandil Brucine-sulphanilic acid in

H2SO4 medium

410 [94]

22

3-Methyl-2-

benzothiazoline hydrazone

HCl-metol

560 [94]

N-(1-Naphthyl)

ethylenediamine

dihydrochloride

525 [95]

Nifedipine KMnO4 in neutral medium 530 [96]

4-Methyl amino phenol

and K2Cr2O7

525 [97]

Bromocresol green

415 [98]

Bromophenol blue

415 [98]

Bromothymol blue

415 [98]

Eriochrome Black T

520 [98]

KOH in

dimethylsulphoxide

430

[99]

Ammonium molybdate 830 [99]

Norfloxacin KMnO4 in alkaline

medium

603 [100]

Pantoprazole

sodium

Potassium ferricyanide and

ammonium ferric sulphate

725 [101]

Perindopril

erbumine

1-Chloro-2,4-

dinitrobenzene in dimethyl

sulphoxide

420

[102]

Zn(II) and eosin 510 [103]

Ramipril Potassium iodate and

potassium iodide in

aqueous medium

352

[104]

1-Chloro- 2,4

dinitrobenzene

420

[105]

23

Silymarin KMnO4 in neutral medium

3-Methyl-2-

benzothiazoline

hydrazone& potassium

persulphate

530

430

[106]

[107]

Trimethoprim Persulphate in alkaline

medium

355 [108]

Nitrous acid 420 [109]

Verapamil HCl Chloramine T 425 [110]

N-Bromosuccinimide 415 [111]

Potassium metaperiodate 425 [112]

Tropaeolin 000 No.1 400 [112]

24

TABLE 1.4: Analytical characteristics of derivative procedures for determination of

pharmaceutical compounds.

Compound Deriv.

Order

λmax/

(nm)

Linear

range/

µg mL-1

Application

remark

Ref.

Doripenem

1st 324 (0.42-11.30)

× 10-2

Degradation

products

[115]

Estradiol valerate 1st 270 200-400 Tablet [116]

Fluocinolone acetonide 2nd 250-400 0.062–0.312

mM

Solvolysis [117]

Galanthamine hydrobromide Zero 287 30-80 bulk and

pharmaceutical

formulation

[118]

1st 277.4 30-80 bulk and

pharmaceutical

formulation

[118]

Hydrochlorothiazide 1st 233 1-6 Tablet [119]

2nd 231 1-6 Tablet

Pseudoephedrine hydrochloride

2nd 271 200 - 1000

Tablet and

Dissolution

testing

[120]

Triamterene 1st 243 0.75-5 Tablet [119]

2nd 224 0.75-5 Tablet

Triprolidine hydrochloride

2nd 321 10-50

Tablet and

Dissolution

testing

[120]

25

Near infrared spectroscopy (NIRS)

Near infrared spectroscopy is a fast and non-destructive technique that offers

many advantages of industrial applications. The near-infrared (NIR) region (780-2500

nm) is situated between the red band of the visible light and the mid infrared (mid-IR)

region. The NIR signal is a consequence of the absorption of light due to molecular

vibrations (overtones and combinations of fundamental vibrations) of hydrogen bonds

like C-H, N-H, O-H. Within the spheres of pharmaceutical sciences, NIR spectroscopy is

now widely accepted as a valuable analytical tool. It can be assumed that the ICH’s

quality of design [121] and the U.S. Food and Drug Administration’s process analytical

technology (PAT) [122] initiatives laid the cornerstone for the increasing interest of the

pharmaceutical industry in NIR spectroscopy. Thorough reviews on the application of

NIR spectroscopy in pharmaceutical development have been published [123-125].

General aspects of NIR analyses are also described in the Ph.Eur [126] and the USP

[127].

Pharmaceutical Analytical Science Group [128] has written an interesting

document, entitled “Guidelines for the development and validation of NIR spectroscopic

methods”. NIR spectroscopy can be applied to the process that can be either at-line, on-

line or in-line [122] and thus able to provide fast, non-destructive and non-invasive

process information. This development also led to a document of the European Medicine

Agency regarding the use of NIR spectroscopy by the pharmaceutical industry [129]. For

fluid bed and high shear processes, NIR has been applied for the investigation of blend

homogeneity, moisture content and process monitoring [130-132]. There are also

numerous publications reporting the application of NIR for quantification of active

pharmaceutical ingredients. NIR spectroscopy has been used to determine the diclofenac

sodium powder with a root mean square error between one and two percent [133].

Recently, a study investigated the quantification of ibuprofen in sustained release

26

formulations and found a root mean square error of 0.85 % after applying both first

derivative and multiplicative scatter correction [134].

Nuclear magnetic resonance (NMR) spectroscopy

The most important role NMR plays in pharmaceutical analysis is its use in

elucidating and for confirming the structures of drug-related substances. However, NMR

is also used to study the drug impurities and contaminants including solvents, synthetic

precursors, synthetic intermediates and decomposition products. In the case of natural

products, NMR may be used to determine the identity of co-extractives. It also has a role

to play in the study of drug metabolism where it has been used for identification and

quantification of many metabolites.

The quantitative analysis by NMR is based on the fact that the intensity, I, of

NMR signal is directly proportional to the number of nuclei, N, evoking the signal. The

linear relationship between intensity of NMR signal and the number of nuclei (in case of

single pulse excitation) is expressed as:

I = Cs × N

where Cs is the proportionality constant arising from the parameters of the spectrometer

and the sample.

Principles and challenges of different NMR methods for studying spectra of

complex mixtures can be found in the literature [135]. Recently comprehensive reviews

on techniques and applications of NMR spectroscopy for pharmaceutical analysis have

appeared [136-139]. Polynuclear NMR consisting of 1H,

13C and

15N has been used for

the analysis of sildefanil base and citrate in solution, solid state and pharmaceutical

dosage forms [140]. Quantitative 1H NMR method has been employed for the

simultaneous analysis of obidoxime chloride and atropine sulphate in parental injection

devices with the use of sodium 3- (trimethyl silyl) - 1- propane sulphonate hydrate as

27

internal standard [141]. It has been demonstrated [142,143] that 2D diffusion–ordered 1H

NMR spectroscopy (2D DOSY 1H NMR) is an interesting method for the complete

characterization of pharmaceutical formulations. The formulations of tadalafil were

analysed by 1H NMR and 2D DOSY

1H NMR spectroscopy [144].

Spectrofluorimetric methods

Fluorescence spectrometry has been firmly established as a sensitive and specific

technique for detection and determination of trace quantities of drugs in pharmaceutical

preparations and biological fluids. The technique involves the measurement of an

enhanced or quenched fluorescent signal of the analyte. Regarding fluorimetric

methodologies, some methods involve chemical reactions while others are based on the

native fluorescence of analytes. One can also distinguish between equilibrium or end

point methods and kinetic methods, the latter include methods involving reaction-rate or

fluorescence life time measurements. Generally a fluorimetric method has the potential of

being 10-100 folds more sensitive than a colorimetric method. Spectrofluorimetry has

been used for determination of active pharmaceutical ingredients in bulk drug and

formulations and details of these methods are recorded in Table 1.5.

Electranalytical methods

Electroanalytical techniques provide high sensitivity with low detection limits and

associated with simple and inexpensive instrumentation. Electrogravimetry, coulometry,

conductometry, polarography, potentiometry, amperometry and voltammetry are

electroanalytical techniques that have been used for the determination of drugs in dosage

forms. Besides their analytical advantages, these techniques play an important role in the

study of pharmacologically active compounds since most of the metabolic pathways

involving these substances are based on redox reactions. However, voltammetric

techniques have been used most commonly for analysis of drugs.

28

Table 1.5: Quantitative analysis of drugs in pharmaceutical formulations by

spectrofluorimetry.

Name of drug Reagents used Λex (nm) / Λem (nm) References

Alprazolam Sodium dodecyl

sulfate (SDS),

metal salt solution

325/520 nm [145]

Antimigrane drugs 4- Chloro- 7-

nitrobenzofurazan

235,460/357,550 nm [146]

Captopril

Cerium(IV)

256 / 354 nm.

[147]

Carvedilol Dansyl chloride 350 / 445 nm [148]

Clomipramine Alizarin red S 490 / 561 nm [149]

Doxepin Hydrochloride Eosin Y 464 / 567 nm [150]

Duloxetine hydrochloride Acetic acid 225 / 340 nm [151]

Fexofenadine hydrochloride Silver nanoparticles 391 / 540 nm [152]

Fluoroquinolones 2,3,5,6-Tetrachloro-

p-benzoquinone

359 -363 / 442 -488 nm.

[153]

Haloperidol Alizarin red S 466 / 564 nm

[154]

Labetalol HCl Eosin Y 317 / 452 nm [155]

Naftidrofuryl oxalate Sulfuric acid 258 / 385 nm [156]

Oxamniquine Dansyl chloride 335 / 445 nm [157]

Paracetamol and mefenamic

acid

Sodium dodesyl

sulfate

405 / 460 [158]

Pregabalin

4-Chloro-7-

nitrobenzofurazan

460 / 558 nm [159]

Tinidazole, Rofecoxib,

Nimesulide

N-

Methylnicotinamide

Chloride (NMNCl)

336,354,325/391,440,375

nm

[160]

29

Comprehensive reviews on the application of these techniques for drug analysis have

appeared in the literature [161-164]. The most commonly used voltammetric techniques

are linear sweep voltammetry (LSV), differential pulse voltammetry (DPV) and square

wave voltammetry (SWV). The sensitivity of voltammetric techniques can be

significantly improved by using a preconcentration step before voltammetric scan. This

procedure is called stripping voltammetric techniques. Several materials can be used as

working electrode in voltammetric techniques and they can be classified into two groups:

(i) Mercury electrodes (dropping mercury electrode, DME, and hanging mercury

drop electrode, HMDE),

(ii) Solid electrodes such as platinum, gold, glassy carbon and carbon paste

electrodes.

Voltammetric techniques are especially useful for drug analyses in dosage forms because

most of the pharmacologically active compounds have electroactive groups. On the other

hand, excipients are ususlly non-electroactive compounds. Therefore, voltammetric

techniques can be directly employed for drug quantification in dosage forms by simply

dissolving the sample in an appropriate solvent without any additional pretreatment. The

application of electroanalytical techniques for quantification of drugs is presented in

Table 1.6.

The present thesis deals with development of new analytical methods for the

determination of Haloperidol, Doxepin hydrochloride, Amitriptyline hydrochloride and

Perindopril erbumine in pharmaceutical preparations.

30

Table 1.6: Electroanalytical methods for analysis of drugs.

Drug Electrode Technique Sample Reference

Cefepime HMDE CV dosage form

and urine

[165]

Cefixime HMDE DP-CAdSV dosage form,

Human urine

[166]

Cetrizine

dihydrochloride

GCE modified

with MWCNT

CV dosage form

and urine

[167]

Ezetimibe GCE modified

with MWCNT

Amperometry dosage form [168]

Gatifloxacin,

moxifloxacin

and sparfloxacin

- Conductometry dosage form [169]

Ofloxacin and

Ornidazole

HMDE LSV dosage form [170]

Rosiglitazone HMDE SW-AAdSV urine, plasma [171]

Vitamin K3 DME DPP dosage form,

serum

[172]

Ziprasidone

hydrochloride

ISE Potentiometry dosage form [173]

GCE-MWCNT = Glassy carbon electrode modified with multi walled carbon nanotube.

SW-AAdSV = Square wave anodic adsorptive stripping voltammetry.

DP-CAdSV = Differential pulse cathodic adsorptive stripping voltammetry.

DPP = Differential pulse polarography.

ISE = Ion selective electrode.

CV = Cyclic voltammetry.

31

HALOPERIDOL

Haloperidol, 4-[4-(4-chlorophenyl)-4- hydroxy-1-piperidinyl]-1-(4-fluorophenyl)

butan-1-one, is a typical butyrophenone antipsychotic drug. It is used in the treatment of

chronic Schizophrenia and acute psychotic states and delirium [174]. It is an odourless

white to pale yellow crystals or white powder with the molecular weight of 375.87.

Haloperidol is practically insoluble in water, slightly soluble in alcohol, methanol and

methylene chloride. It is commercially available as tablets and injections. The

recommended dose varies based on the indication, usually found to be 5-15 mg per day

with an average of 10 mg per day. Haloperidol has a pKa value of 8.3. It should be stored

in tight and light resistant containers. Haloperidol is metabolized to several metabolites

(Fig. 1.4) and the enzyme involved in the biotransformation of haloperidol include

cytochrome P450, carbonyl reductase and uridine diphosphoglucose glucuronosyl

transferase [175]. The greatest proportion of the intrinsic hepatic clearance of haloperidol

is by glucuronidation followed by the reduction of haloperidol to reduced haloperidol and

by cytochrome P450-mediated oxidation [176].

The drug is noted for its strong early and late extrapyramidal side effects. The risk

of facial disfiguring tardive dyskinesia is around 4 % per year in younger patients. In the

patients over the age of 45, the percentage of those afflicted can be even higher. Other

side effects include dry mouth, lethargy, restlessness of akathisia, muscle-stiffness,

muscle-cramping and weight gain. Such side effects occur more likely when drug is

given in high doses and/or during long term treatment.

32

F C

OH

CH2 CH2 CH2 N

Cl

OH

Reduced haloperidol

F C

O

CH2 CH2 CH2 N

Cl

OH

Haloperidol

F C

O

CH2 CH2 CO2H HN

Cl

OH

4-Fluorobenzoylpropionic acid Piperidine metabolite

Fig. 1.4. Metabolism of haloperidol.

33

DOXEPIN HYDROCHLORIDE

Doxepin hydrochloride, chemically known as 3-dibenz [b,e] oxepin-11 (6H)

ylidene-N, N-dimethyl propan-1-amine hydrochloride (CAS: 1229-29-4, M. W. 315.8), is

a tricyclic antidepressant which displays a potent central anticholinergic activity and can

alter the activity of both noradrenergic and serotonergic pathways. It is administered as

an 85.15 % mixture of the E-Z stereoisomers. Fig. 1.5 shows the structures of E- and Z-

doxepin. The E- and Z- isomers of doxepin have different potencies in the central nervous

system; in all animal models of depression Z- doxepin is more active than E- doxepin

[177].

O

N

CH3

H3C

O

N

CH3

CH3

E-Doxepin Z-Doxepin

Fig. 1.5. E- and Z- Doxepin.

It is a white crystalline powder, freely soluble in water, alcohol and methylene chloride. It

is most widely used for the treatment of depression, anxiety and sleep disorders. The

recommended dosage varies based on the indication, usually ranging from 75 mg/day to

150 mg/day. However, at high concentrations severe adverse effects and toxicity can

appear like cardiac arrhythmias and severe hypotension. Its principal oxidative

metabolites detected in human urine [178] are (E) - 2- hydroxydoxepin, (E)-2-hydroxy-

N-desmethyldoxepin, (Z)-N-desmethyldoxepin and (E)-N-desmethyldoxepin and (Z)-

doxepin-N-oxide and (E)-doxepin-N-oxide. The metabolite N- desmethyldoxepin is also

pharmacologically active and has greater sedative properties than the parent drug.

34

AMITRIPTYLINE HYDROCHLORIDE

Amitriptyline is a tricyclic antidepressant with sedative properties. It is chemically

described as {3-(10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5-ylidene)-N,N-dimethyl-1-

propanamine hydrochloride}. The steps involved (Fig. 1.6) in the synthesis of

amitriptyline are:

(i) cyclization of dibenzyl-o-carboxylic acid (I) by means of polyphosphoric acid at

170°C leads to 10, 11-dihydro-5H-dibenzo [a, d] cyclohepten-5-one (II);

(ii) the treatment of II with 3-dimethylamino propyl magnesium yields the corresponding

tertiary carbinol (III);

(iii) the dehydration of III with acetic anhydride or acetyl chloride in chloroform results

in amitriptyline (IV).

Amitriptyline hydrochloride [CAS No. 549-18-8] is a white, colourless, crystalline

compound with the molecular weight of 313.87. It is freely soluble in water. Chemically

it is basic (log k of 9.42), and hydrophobic (logPo/w of 4.64). Amitriptyline is metabolized

mainly by demethylation forming nortriptyline (Fig. 1.7), and by hydroxylation, leading

to the formation of E-10-hydroxy (EHAT) and Z-10-hydroxyamitriptyline (ZHAT).

Nortriptyline is further demethylated to desmethylnortriptyline (NNT) and hydroxylated

to E-10-hydroxy nortriptyline (EHNT) and Z-10-hydroxynortriptyline (ZHNT). The

demethylation of amitriptyline and nortriptyline is mainly catalysed by CYP2C19, with

the participation of other CYP enzyme forms in higher drug concentrations. The

formation of E-10-hydroxy metabolites is dependent on the activity of CYP2D6, with

stereospecificity to the (-)-EHAT and (-)-EHNT metabolites.

35

Fig. 1.6. Synthesis of amitriptyline.

NCH3

Amitriptyline

CYP2D6

CYP2C19

N

CH3

EHATCH3

CH3

NH3C

H3C

ZHAT

CYP2C19

CYP2C19

NCH3

H

Nortriptyline

CYP2D6

N

CH3

HEHNT

NH3C

H3C

ZHNT

CYP2C19

NH

H

NNT

HO

HOHO

HO

Fig. 1.7. Main metabolite pathways of amitriptyline.

36

. Pharmacokinetics of this drug is related to absorption and elimination in plasma.

Peak plasma concentration occurs within 2 to 12 h after oral administration. The plasma

half-life of amitriptyline ranges from 10 to 50 h. Doses of upto 200 mg daily to 300 mg

daily have been used in severely depressed patients. Approximately 25-50 % of a dose is

excreted in urine as inactive metabolites within 24 h, and small amounts are excreted in

faeces via biliary elimination.

PERINDOPRIL ERBUMINE

Perindopril erbumine is a new member of angiotensin-converting enzyme

inhibitors group. It is chemically known as (2S, 3aS, 7aS)-1-[(S)-N-[(S)-1-carboxybutyl]

alanyl] hexahydro-2-indolinecarboxylic acid, 1-ethylester, compound with tert-

butylamine (1:1). It is a white crystalline powder which is freely soluble in water, alcohol

and chloroform. Perindopril is a pro drug and metabolized in vivo by hydrolysis of the

ester group to form perindoprilat, the biologically active metabolite. It is safely

administered for the treatment of hypertension [179]. According to the dose response

studies [180], the recommended dose to be used in hypertensive patients is 4 mg once a

day that could be increased to 8 mg once a day if necessary. The commercial tablets

contain 2 mg, 4 mg or 8 mg of perindopril for oral administration. In addition to

perindopril erbumine, each tablet contains the inactive ingredients such as colloidal silica,

lactose, magnesium stearate and microcrystalline cellulose. Following oral

administration, perindopril is rapidly absorbed and metabolized; only 4-12 % of the dose

is recovered unchanged in urine. The degradation products [181] of perindopril erbumine

include perindoprilat, Y31. Y32, and Y33, but they can also be present as impurities from

the synthesis route. The structural formulae and IUPAC names of these impurities are

given below (Fig. 1.8):

37

H3C O

H3C N

O

H

CH3

O

NH

H

COOH,

(9)(11)

(7a) (2)

NH2

CH3

CH3

3(a)

Perindopril erbumine

(2a,3aS,7aS)-1-((S)-N-((S)-1-(ethoxycarbonyl)alanyl)octahydro-1H-indole-2-carboxylic acid,ter-butylamine salt

N

O

N

O

O

O CH3

CH3

CH3H

H

N

O

N

O

O

OH

CH3

CH3H

H

H

Impurity Y 31

H

Impurity Y 32

N

O

N

O

O

OH

CH3

CH3H

H H

Impurity Y 33

((2S)-2-((2S,4aS,5aS,9aS)-2-methyl-1,4-dioxooctahydro-1H-indolo(1,2-a)piperazin-3-yl)pentanoic acid

((2S)-2-((2S,4aR,5aS,9aS)-2-methyl-1,4-dioxooctahydro-1H-indolo(1,2-a)piperazin-3yl) pentanoic acid

((2S)-ethyl(2-((2S,4aS,5aS,9aS)-2-methyl-1,4-dioxooctahydro-1H-indolo(1,2-a)piperazin-3yl)pentanoate

CH3

Fig. 1.8. Structures of perindopril erbumine and its impurities.

38

REFERENCES

[1] H.J. Barber, Historical aspects of chemotherapy, May and Baker Ltd., (1978),

Dagenham, UK.

[2] O. Shakoor, R.B. Taylor and R.R. Moody, Analyst 120 (1995) 2191.

[3] S. Foster, Soc. Sci. Med. 32 (1991) 1201.

[4] M.T. Ham, Adverse drug React. Toxicol. Rev. 11 (1992) 59.

[5] S. K. Pandya, Br. Med. J. 297 (1988) 117.

[6] M.M. Silverman, M. Lydecker and P.R. Lee, Int. J. Health Services 20 (1990)

561.

[7] International Conference on Harmonization (2000) Draft Revised Guidance On

Impurities In New Drug Substances. Federal Register Q3A(R) 65 (140): 45085.

[8] International Conference on Harmonization (2000) Draft Revised Guidance On

Impurities In New Drug Products. Federal Register Q3B(R) 65 (139) 44791.

[9] International Conference on Harmonization (1997) Impurities, Q3C Guidelines

for Residual Solvents, Q3C. Federal Register 62 (247) 67377.

[10] International Conference on Harmonization (1999) Specifications, Q6A: Test

Procedures and Acceptance Criteria for New Drug Substances and New Drug

Products. Chemical substances 65 (146) 67488.

[11] S. Ahuja, Impurities Evaluation of Pharmaceuticals. Marcel Dekker, New York,

(1998), p. 142.

[12] T.N. Riley, Steric aspects of drug action. Pharmacist 23 (1998) 40.

[13] International Conference on Harmonization (1997) Impurities, Q3C Guidelines

for Residual Solvents, Q3C. Federal Register 62 (247) 67377.

[14] F.M. Dayril and M.C. Dumlao, Forensic Sci. Int. 144 (2004) 29.

39

[15] V. Buhler, Vademecum for Vitamin Formulation. Stuttgart, Germany, Wiss,

Verl-Ges., 142 (1998) 36.

[16] J. Roy, M. Islam, A.H. Khan, S.C. Das, M. Akhteruzzaman, A.K. Deb and A.H.

Alam, J. Pharma. Sci. 90 (2001) 541.

[17] J. Roy, M. Mahmud, A. Sobhan, M. Aktheruzzaman, M. Al-Faooque and E. Ali,

Drug Dev. Ind. Pharm. 20 (1994) 2157.

[18] S.L. Hoerle, K.D. Evans and B.G. Snider, HPLC Determination of Impurities in a

3rd Generation Cephalosporine, Eastern Analytical Symposium, November 16-

20, Somerset, New Jersey, (1992), p. 12.

[19] M. Gazdag, M. Babjag, J. Brlik, S. Maho, Z. Tuba and S. Gorog, J. Pharm.

Biomed. Anal. 17 (1998) 1029.

[20] A. Smith, P.M. Pennefather, S.B. Kaye and C.A. Hart, Indian Drugs 61 (2001)

747.

[21] N.R. Rao, S.S. M. Kiran and N.L. Prasanthi, Indian J. Pharm. Educ. Res. 44

(2010) 301.

[22] K. Valko (ed), In: Handbook of analytical separation, vol. 1. Elsevier,

Amsterdam, (2000).

[23] J. Pawliszyn (ed), In: Comprehensive analytical chemistry, vol XXXVII. Elsevier,

Amsterdam, (2002).

[24] D.A. Wells In: Progress in pharmaceutical and biomedical analysis, vol. 5.

Elsevier, Amsterdam, (2003).

[25] G. Hempel (ed), In: Handbook of analytical separations vol 5. Elsevier,

Amsterdam, (2004).

[26] The European Pharmacopoeia, 4th

edn., Council of Europe, Strasbourg, (2002).

40

[27] United States Pharmacopoeia, 27th

edn., The USP Convention Inc., Rockville,

MD (2004).

[28] N.R. Prasad, Chemical industry and chemical engineering quarterly 17 (2011) 99.

[29] K.B. Vinay, H.D. Revanasiddappa, N. Rajendraprasad, M.S. Raghu, P.J. Ramesh,

C.M. Xavier and K. Basavaiah, J. Preclin. Clin. Res. 4 (2010) 019.

[30] A. Joseph and A. Rustum, J. Pharm. Biomed. Anal. 51(2010) 521.

[31] M.J. Skibic, L.A. King, M. Khan, P.J. Fox, B.E. Winger and S.W. Baertschi, J.

Pharm. Biomed. Anal. 53 (2010) 432.

[32] M.S. Elmasry, I.S. Blagbrough, M.G. Rowan, H.M. Saleh, A.A. Kheir and P.J.

Rogers, J. Pharm. Biomed. Anal. 54 (2011) 646.

[33] V. K. Chakravarthy and D.G. Shankar, Rasayan J. Chem. 4 (2011)165.

[34] J.W. Collier, R.B. Shah, A.R. Bryant, M.J. Habib, M.A. Khan and P.J. Faustino,

J. Pharm. Biomed. Anal. 54 (2011) 433.

[35] I.I. Salem, M. Alkhatib and N. Najib, J. Pharm. Biomed. Anal. Article in press

(2011), doi:10.1016/j.jpba.2011.07.020

[36] L. Chytil, B. Strauch, J. Cvacka, V. Maresova, J.W. Jr, R. Holaj and O. Slanar, J.

chromatogr. B 878 (2010) 3167.

[37] Yan-Hui Wang, C. Qiu, Da-Wei Wang, Zheng-Fang Hu, Bo-Yang Yu and Dan-

Ni Zhu, J. Pharm. Biomed. Anal. 54 (2011) 1110.

[38] S. Geenen, F. Michopoulos, J.G. Kenna, K.L. Kolaja, H.V. Westerhoff and I.

Wilson, J. Pharm. Biomed. Anal. 54 (2011) 1128.

[39] A. D’Avolio, M. Simiele, M. Siccardi, L. Baietto, M. Sciandra, V. Oddone, F.R.

Stefani, S. Agati, J. Cusato, S. Bonora and G.D. Perri. J. Pharm. Biomed. Anal. 54

(2011) 779.

[40] J. He, T. Qin, J. Wen, F. Qin and F. Li, J. Pharm. Biomed. Anal. 54 (2011) 551.

41

[41] M. Indupriya, R.S. Chandan, B.M. Gurupadayya and K. Sowjanya, Int. J. Pharm.

Tech. 3 (2011) 1694.

[42] A. Subasranjan, P. Suresh, C. Srinivasulu and R. Hemant, Int. J. Pharm. Tech. 2

(2010) 182.

[43] H. Liu, Q. Tang, R.J. Markovich and A.M. Rustum, J. Pharm. Biomed. Anal. 54

(2011) 417.

[44] G. Szepesi and S. Nyiredy, Pharmaceutical and drugs. in: J. Sherma, B. Fried

(Eds.). Handbook of Thin-Layer chromatography, 2nd

edn., Marcel Dekker, New

York, (1996) p. 208.

[45] K. Ferenczi-Fodor, Z. Vegh and B. Renger, J. Chromatogr. A 1218 (2011) 2722.

[46] J.M. Płotka, C. Morrison and M. Biziuk, Trends Anal. Chem. 30 (2011) 1139.

[47] V.P. Choudhari1 and A.P. Nikalje, Chromatographia 70 (2009) 309.

[48] H.N. Dave, R.C. Mashru and A.K. Patel, J. Pharm. Sci. Res. 2 (2010) 143.

[49] M. Jelen, B. Morak-Mlodawska and K. Pluta, J. Pharm. Biomed. Anal. 55 (2011)

466.

[50] S.M. Pawar, B.S. Patil and R.Y. Patil, Int. J. Adv. Pharm. Sci. 1 (2010) 52.

[51] D.H. Shewiyo, E. Kaale, C. Ugullum, M.N. Sigonda, P.G. Risha, B. Dejaegher, J.

Smeyers–Verbeke and Y. Vander Heyden , J. Pharm. Biomed. Anal. 54 (2011)

445.

[52] D.C. Oliva, K.T. Velez and A.L.R. Vazquez, J. Mex. Chem. Soc. 55 (2011) 79.

[53] Yu-Shan Huang, Shun-Niang Chen andChen-Wen Whang, Electrophoresis 32

(2011) 2155.

[54] U. Franzen, C. Vermehren, H. Jensen and J. Ostergaard, Electrophoresis 32

(2011) 738.

[55] F.S. Felix, C.M.A. Brett and L. Angnes, Talanta 78 (2008) 128.

42

[56] D. Yeniceli, D. Dogrukol-Ak and M. Tuncel, J. Pharm. Biomed. Anal. 36 (2004)

145.

[57] A.F. Lavorante, C.K. Pires and B.F. Reis, J. Pharm. Biomed. Anal. 42 (2006) 423.

[58] H.P.A. Nouws, C. Delerue-Matos, A.A. Barros, J.A. Rodriguez and A. Santos-

Silva, Anal. Bioanal. Chem. 382 (2005) 1662.

[59] P. Norouzi, M.R. Ganjali and P. Matloobi, Electrochem. Commun. 7 (2005) 333.

[60] I.F. Al-Momani, A.T. Haj-Hussein and A.N. Tahtamouni, J. Flow Injection Anal.

25 (2008) 151.

[61] J. Ruzicka and G.D. Marshall, Anal. Chim. Acta 237 (1990) 329.

[62] Y. M. Issa and A.F. Khorshid, J. Adv. Res. 2 (2011) 25.

[63] P. Vinas, C. Lopez-Erroz, F.J. Cerdan, N. Campillo and M. Hermandez-Cordoba,

Mikrochim. Acta 134 (2000) 83.

[64] S.Z. Qureshi, A. Saeed and N. Rahman, Chem. Anal. (Warsaw) 37 (1992) 227.

[65] N. Rahman, N.A. Khan and S.N.H. Azmi, Anal. Sci. 20 (2004) 1231.

[66] N. Rahman and S.N.H. Azmi, Anal. Sci. 16 (2000) 1353.

[67] N. Rahman and S.N. H. Azmi, IL Farmaco 56 (2001) 731.

[68] N. Rahman and M.N. Hoda, J. Pharm. Biomed. Anal. 31 (2003) 381.

[69] M.I.H. Helaleh, N. Rahman and R.M.A. Q. Jamhour, Chem. Anal. (Warsaw)

42 (1997) 261.

[70] A.S. Ahmad, N. Rahman and F. Islam, J. Anal. Chem. 59 (2004) 119.

[71] S.Z. Qureshi, A. Saeed, S. Haq and N. Rahman, Anal. Lett. 23 (1990) 995.

[72] N. Rahman and S.N.H. Azmi, Microchem. J. 65 (2000) 39.

[73] N. Rahman and S.N.H.Azmi, J. Pharm. Biomed. Anal. 24 (2000) 33.

[74] N. Rahman and S.M. Haque, Anal. Chem. Insights 3 (2008) 31.

[75] N. Rahman, Z. Bano and S.N.H. Azmi, J. Chin. Chem. Soc. 55 (2008) 557.

43

[76] N. Rahman and M. Kashif, Anal. Sci. 19 (2003) 907.

[77] N. Rahman and M. Kashif, IL Farmaco 58 (2003) 1045.

[78] N. Rahman, M.R. Siddiqui and S.N.H. Azmi, Chem. Pharm. Bull. 54 (2006) 626.

[79] N. Rahman and N.Afaq, Drug testing and Anal. 2 (2010) 442.

[80] M.I.H. Helaleh, N. Rahman, R.M.A.Q. Jamhour and E.S.M.A. Nameh, J. Pharm.

Biomed. Anal. 16 (1997) 269.

[81] N. Rahman, M. Singh and M.N. Hoda, Chin. J. Chem. 23 (2005) 1.

[82] N. Rahman, H. Rahman and S.N.H. Azmi, J. Chin. Chem. Soc. 54 (2007) 185.

[83] N. Rahman, N. Anwar, M. Kashif, M.M. Hoda and H. Rahman, J. Mex. Chem.

Soc. 55 (2011) 105.

[84] M.I.H. Helaleh, N. Rahman and E.S.M.A. Nameh, Anal. Sci.13 (1997) 1007.

[85] N. Rahamn, N. Anwar and M. Kashif, IL Farmaco 60 (2005) 605.

[86] N. Rahman, M. Singh and M.N. Hoda, J. Braz. Chem. Soc. 16 (2005) 1001.

[87] N. Rahman, M.R. Siddiqui and S.N.H. Azmi, Chem. Anal. (Warsaw) 52(2007)

465.

[88] N. Rahman, M.R. Siddiqui and S.N.H. Azmi, J. Chin. Chem. Soc. 53 (2006) 735.

[89] M.I.H. Helaleh, N. Rahman and R.M.A.Q. Jamhour, Annali di chim. (Rome) 86

(1996) 509.

[90] N. Rahman, Y. Ahmad and S.N.H. Azmi, Chem. Anal. 50 (2005) 769.

[91] N. Rahman, H. Rahman and S.N.H. Azmi, Chem. Pharm. Bull. 53 (2005) 942.

[92] N. Rahman, Y. Ahmad and S. N. H. Azmi, Chem. Anal. 50 (2005) 769.

[93] M. I. H. Helaleh, S. Z. Qureshi, N. Rahman and R. M. A. Q. Jamhour, Acta Pol.

Pharm. Drug Research 54 (1997) 111.

[94] N. Rahman, N.A. Khan and S.N.H Azmi, IL Farmaco 59 (2004) 519.

[95] N. Rahman, M.R. Siddiqui and S.N.H. Azmi, Yakugaku Zasshi 127 (2007) 367.

44

[96] N. Rahman and S.N.H. Azmi, Acta Pharm. (Zagreb) 49 (1999) 113.

[97] N. Rahman and M.N. Hoda, IL Farmaco 57 (2002) 435.

[98] N. Rahman, N.A. Khan and S.N.H. Azmi, IL Farmaco 59 (2004) 47.

[99] N. Rahman and S.N.H. Azmi, Acta Biochim. Pol. 52 (2005) 915.

[100] N. Rahman, Y. Ahmad and S.N.H. Azmi, Eur. J. Pharm. Biopharm. 57 (2004)

359.

[101] N. Rahman, Z. Bano and S.N.H. Azmi, Anal. Sci. 22 (2006) 983.

[102] N. Rahman, N. Anwar and M. Kashif, Chem. Pharm. Bull. 54 (2006) 33.

[103] N. Rahman and H. Rahman, Spectroscopy 25 (2011) 123.

[104] N. Rahman, Y. Ahmad and S.N.H. Azmi, AAPS Pharm. Sci. Tech. 06 (2005) 543.

[105] N. Rahman, H. Rahman and S.N.H. Azmi, Inter. J. Pharm. Biomed. Sci. 2 (2007)

52.

[106] N. Rahman, N.A. Khan and S.N.H. Azmi, Pharmazie 59 (2004) 112.

[107] N. Rahman, Y. Ahmad and S.N.H. Azmi, Can. J. Anal. Sci. Spec. 50 (2005) 116.

[108] S.Z. Qureshi, M.I.H. Helaleh, N. Rahman and R.M.A.Q. Jamhour, Fresenius J.

Anal. Chem. 357 (1997) 1005.

[109] S.Z. Qureshi, M.I.H. Helaleh, N. Rahman and, R.M.A.Q. Jamhour, Chem. Anal.

(Warsaw) 42 (1997) 65.

[110] N. Rahman and M.N. Hoda, Anal. Bioanal. Chem. 374 (2002) 484.

[111] N. Rahman and S.N.H. Azmi, IL Farmaco 59 (2004) 529.

[112] N. Rahman, N.A. Khan and S.N.H. Azmi, Sci. Asia 31(2005) 341.

[113] T.C.O. Haver, Anal. Chem. 51 (1979) 91 A.

[114] A.F. Fell, Quality control of Steroids in Pharmaceutical Formulations by Higher

Derivative Spectroscopy, in Advances in Steroids Analysis, S. Gorog, (Ed.);

Elsevier, Amsterdam, (1982), p. 495.

45

[115] J. Cielecka-Piontek and A. Jelinska, Spectrochim. Acta A 77 (2010) 554.

[116] A.S.L. Mendez, L. Deconto and C.V. Garcia, Quim. Nova 33 (2010) 981.

[117] B. Markovic, S. Vladimirov, O. Cudina, V. Savic and K. Karljikovic-Rajic,

Spectrochim. Acta A 75 (2010) 930.

[118] K. Mittal1, R. Kaushal, R. Mashru and A. Thakkar, J. Biomed. Sci. Eng. 3 (2010)

439.

[119] K. Mohammadpour, M.R. Sohrabi and A. Jourabchi, Talanta 81 (2010) 1821.

[120] L. Sriphong, A. Chaidedgumjorn, K. Chaisuroj, World Academy of Science,

Engineering and Technology 55 (2009) 573.

[121] International Conference on Harmonization of Technical Requirements for

registration of Pharmaceuticals for Human Use 2009: Topic Q8 (R2):

Pharmaceutical Development.

[122] U.S. Food and Drug Administration 2004: Guidance for industry. PAT- A frame

work for Innovative Pharmaceutical Development, Manufacturing, and Quality

Assurance.

[123] M. Blanco, J. Coello, H. Iturriaga, S. Maspoch and C. de la Pezuela, Analyst 123

(1998) 135 R.

[124] E. Rasanen and N. Sandler, J. Pharm. Pharmacol. 59 (2007) 147.

[125] J. Luypaert, D.I. Massart and Y.V. Heyden, Talanta 72 (2007) 865.

[126] The Eurapean Pharmacopoeia, 5th

edn., Council of Europe, Strasbourg (2004).

[127] The United States Pharmacopoeia, 26th

edn., NF 22 (2003).

[128] Pharmaceutical Analytical Science Group, NIR sub Group, Guidelines for the

development and validation of Near Infrared Spectroscopic methods,

(http://www.pasg.org.uk/NIR/NIR) Guidelines Oct. 01. Pdf (2001).

46

[129] European Medicines Agency 2009: Draft Guideline on the use of Near Infrared

spectroscopy by the pharmaceutical Industry and the Data Requirements for New

submissions and Variations revision 1.

[130] Y. Sulub, B. Wabuyele, P. Gargiulo, J. Pazdan, J. Cheney, J. Berry, A. Gupta, R.

Shah, H.Q. Wu and M. Khan, J. Pharm. Biomed. Anal. 49 (2009) 48.

[131] Y. Zheng, X. Lai, S.W. Bruun, H. Ipsen, J.N. Larsen, H. Lowenstein, I.

Sondergaard and S. Jacobsen, J. Pharm. Biomed. Anal. 46 (2008) 592.

[132] A.U. Vanarase, M. Alcala, J. Rozo, F.J. Muzzio and R.J. Romanach, Chem. Eng.

Sci. 65 (2010) 5728.

[133] B. Whang, G.L. Liu, Y. Dou, L.W. Liang, H.T. Zhang and Y.L. Ren, J. Pharm.

Biomed. Anal. 50 (2009) 158.

[134] X.L. Wang, Q.A. Fu, J.F. Shang, X. Yang, J.Z. Jia and W. Du, Vib. Spectros. 53

(2010) 214.

[135] R. Novoa-Carballal, E. Fernandez-Megia, C. Jimenez and R. Riguera, Nat. Prod.

Rep. 28 (2011) 78.

[136] U. Holzgrabe, Prog. Nucl. Mag. Res. Spectros. 57 (2010) 229.

[137] M. Malet-Matrino and U. Holzgrabe, J. Pharm. Biomed. Anal. 55 (2011) 1.

[138] U. Holzgrabe and M. Malet-Martino, J. Pharm. Biomed. Anal. 55 (2011) 679.

[139] H. Therien-Aubin and X.X. Zhu, Carbohydrate Polymers 75 (2009) 369.

[140] I. Wawer, M. Pisklak and Z. Chilmonczyk, J. Pharm. Biomed. Anal. 38 (2005)

865.

[141] R. Sharma, P.K. Gupta, A. Mazumder, D.K. Dubey, K. Ganesen and R.

Vijayaraghavan, J. Pharm. Biomed. Anal. 49 (2009) 1092.

[142] S. Trefi, V. Gilard, M. Malet-Martino and R. Martino, J. Pharm. Biomed. Anal.

44 (2007) 743.

47

[143] S. Trefi, V. Gilard, S. Balayssac, M. Marlet-Martino and R. Martino, J. Pharm.

Biomed. Anal. 46 (2008) 707.

[144] S. Trefi, C. Routaboul, S. Hamieh, V. Gilard, M. Malet-Martino and R. Martino,

J. Pharm. Biomed. Anal. 47 (2008) 103.

[145] A. Mohd, A.A.P. Khan, S. Bano and K.S. Siddiqi, Arabian J. Chem. Article in

press, doi: 10. 1016/j. arabjc. 2010. 10. 013.

[146] R.I. EI-Bagary, N.G. Mohammed and H.A. Nasr, J. Chem. Pharm. Res. 3 (2011)

304.

[147] A.M. El-Didamony, J. Chin. Chem. Soc. 56 (2009) 755.

[148] L. El Sayed, A. F.T.A. Mohamed and E.A. Taha, Chemical Industry & Chemical

Engineering Quarterly 16 (2010) 31.

[149] N. Rahman and N. Afaq, Anal. Methods 2 (2010) 513.

[150] N. Rahman, S. Siddiqui and S.N.H. Azmi, AAPS Pharm. Sci. Tech. 10 (2009)

1381.

[151] S.L. Prabhu, S. Shahnawaz, C.D. Kumar and A. Shirwaikar, Indian J. Pharm. Sci.

70 (2008) 502.

[152] Z.A. Alothman, N. Bukhari, S. Haider, S.M. Wabaidur and A.A. Alwarthan,

Arabian J. Chem. 3 (2010) 251.

[153] S.T. Ulu, Spectrochim. Acta A 72 (2009) 1038.

[154] N. Rahman and S. Siddiqui, Drug Testing and Anal. 2 (2010) 252.

[155] N. Rahman and S.M. Haque, Int. J. Biomed. Sci. 4 (2008) 100.

[156] S.M. Sabry, T.S. Belal, M.H. Barary and M.E.A. Ibrahim, Int. J. Biomed. Sci. 5

(2009) 283.

[157] N. El-Enany, F. Belal and M. Rizk, J. Fluoresc. 18 (2008) 349.

48

[158] T. Madrakian, A. Afkhami and M. Mohammadnejad, Anal. Chim. Acta 645

(2009) 25.

[159] O.O. Sagirli, Spectrochim. Acta A 72 (2009) 68.

[160] K.M. Elokely, M.A. Eldawy, M.A. Elkersh and T.F. EI-Moselhy, Int. J. Anal.

Chem. DOI: 10.1155/2011/840178.

[161] A. L. Santos, R.M. Takeuchi and N.R. Stradiotto, Current Pharm. Anal. 5 (2009)

69.

[162] Q. Xu, A. Yuan, R. Zhang, X. Biang and D. Chem, X. Hu, Current Pharm. Anal.

5 (2009) 144.

[163] N.A. El-Maali, Bioelectrochemistry 64 (2004) 99.

[164] S.A. Ozkan, B. Uslu and H.Y. Aboul-Enein, Crit. Rev. Anal. Chem. 33 (2003)

155.

[165] K. Balaji, K. Reddaiah, T.M. Reddy and S.R.J. Reddy, Portugaliae Electrochim.

Acta, 29 (20011) 177.

[166] R. Jain, V.K. Gupta, N. Jadon and K. Radhapyari, Anal. Biochem. 407 (2010) 79.

[167] R.H. Patil, R.N. Hegde and S.T. Nandibewoor, Colloids Surf. B: Biointerfaces 83

(2011) 133.

[168] F. Jalali, E. Ahmadi, M. Roushani, Gh. Bahrami and M. Shamsipur, Anal. Lett. 43

(2010) 1481.

[169] S.M. Al-Ghannam, Spectrochim. Acta A 69 (2008) 1188.

[170] P.V. Rege and P.A. Sathe, Int. J. Pharm. Sci. Res. 2 (2011) 2226.

[171] A.F. Al-Ghamdi and M.M. Hefnawy, Arabian J. Chem. In Press (2011), doi:

10.1016/J. arabjc. 2011.07.011.

[172] U.T. Yilmaz and G. Somer, Turk. J. Chem. 35 (2011) 201.

[173] V.K. Gupta, S. Agarwal and B. Singhal, Int. J. Electrochem. Sci. 6 (2011) 3036.

49

[174] B. Santamaria, M. Perez, D. Montero, M. Madurga and F.J. de Abajo, Eur.

Psychiatry 17(2002) 471.

[175] J. Fang and J.W. Gorrod, J. Chromatogr. A 614 (1993) 614.

[176] S. Kudo and T. Ishizaki, Clin. Pharmacokinetics 37 (1999) 435.

[177] R.M. Pinder, R.N. Brogden, T.M. Speight and G.S. Avery, Drugs 13 (1997) 161.

[178] Y.Z. Shu, J.W. Hubbard, J.K. Cooper, G. Mc Kay, E.D. Korchinski, R. Kumar

and K.K. Midha, Drug Metab. Dispos. 18 (1990) 735.

[179] D. Herpin, J.P. Santoni, F. Pouyallon and J. Demange, Current Ther. Res. 45

(1989) 576.

[180] R. Luccioni, Y. Frances, R. Gass, C. Schwab, J.P. Santoni and L. Perret, Eur.

Heart J. 9 (1988) 1131.

[181] M. Medenica, D. Ivanovic, M. Maskovic, B. Jancic and A. Malenovic, J. Pharm.

Biomed. Anal. 44 (2007) 1087.