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… M. Pharm. (Quality Assurance) Sem.III (2010), Dissertation Synopsis

CONTENTS

CHAPTER TITLE PAGE NO.

1 Introduction 1-9

1.1 Analytical chemistry 1

1.2 Analytical development 1-6

1.3 Validation 6-7

1.4 System suitability test 7-8

1.5 Drug profile 8-9

2 Literature review 8-14

3 Aim and objective of study 15

4 Plan of work 16

5 References 17-18

1. INTRODUCTION

1 Gyan Vihar School of Pharmacy


1.1 ANALYTICAL CHEMISTRY

Analytical chemistry is a branch of chemistry that deals with the separation, identification

and determination of components in a sample. It is the science of making quantitative

measurements, which requires background knowledge of chemical and physical concepts.

The use of instrument is an exciting and fascinating part of chemical analysis that interacts

with all areas of chemistry and with many other areas of pure and applied science. Analytical

instrument plays an important role in the production and evaluation of new products,

protection of consumers and environment; it also provides the lower detection limits required

to assure safe foods, water and air.

1.2 ANALYTICAL DEVELOPMENT

Method development is done

1) For new products

2) For existing products

Methods are developed for new products when no official methods are available. Alternate

methods for existing (non-pharmacopoeial) products are developed to reduce the cost and

time for better precision and ruggedness. Trial runs are conducted, method is optimized and

validated. When alternate method proposed is intended to replace the existing procedure

comparative laboratory data including merit/demerits are made available.

Selection of analytical method

The following analytical techniques are usually employed for estimations of different

components in formulations:-

1) Titrimetric and gravimetric

2) Ultraviolet and visible spectrophotometry.

3) Thin layer chromatography

4) High performance liquid chromatography (HPLC)

5) Gas Chromatography (GC)

6) Atomic absorption spectrometry (AAS)

7) Infra-Red absorption spectrophotometry.

Steps of method development

Documentation starts at the very beginning of the development process, a system for full

documentation of the development studies must be established. All data relating to these

studies must be recorded in laboratory notebook or an electronic database.

1. Analyte standard characterization



a) All known information about the analyte and its structure is collected i.e., physical and

chemical properties, toxicity, purity, hygroscopic nature, solubility and stability.

b) The standard analyte (100% purity) is obtained. Necessary arrangement is made for the

proper storage (refrigerator, desiccators and freezer).

c) When multiple components are to be analyzed in the sample matrix, the number of

components is noted, data is assembled and the availability of standards for each one is

determined.

d) Only those methods (MS, GC, HPLC etc.,) that are compatible with sample stability are

considered.

2. Method requirements

The goals or requirements of the analytical method that need to be developed are considered

and the analytical figures of merit are defined. The required detection limits, selectivity,

linearity, range, accuracy and precision are defined.

3. Literature search and prior methodology

The literature for all types of information related to the analyte is surveyed. For synthesis,

physical and chemical properties, solubility and relevant analytical methods. Books,

periodicals, chemical manufacturers and regulatory agency compendia such as USP / NF,

AOAC and ASTM publications are reviewed. Chemical Abstracts Service (CAS) automated

computerized literature searches are convenient.

4. Choosing a method

a) Using the information in the literatures and prints, methodology is adapted. The methods

are modified wherever necessary. Sometimes it is necessary to acquire additional

instrumentation to reproduce, modify, improve or validate existing methods for in-house

analytes and samples.

b) If there is no prior methods for the analyte in the literature, from analogy, the compounds

that are similar in structure and chemical properties are investigated and are worked out.

There is usually one compound for which analytical method already exist that is similar to the

analyte of interest.

5. Instrumental setup and initial studies

a) The required instrumentation is setup. Installation, operational and performance

qualification of instrumentation using laboratory standard operating procedures (SOP’s) are

verified.

b) Always new consumables (e.g. solvents, filters and gases) are used, for example, method

development is never started, on a HPLC column that has been used earlier.



c) The analyte standard in a suitable injection / introduction solution and in known

concentrations and solvents are prepared. It is important to start with an authentic, known

standard rather than with a complex sample matrix. If the sample is extremely close to the

standard (e.g., bulk drug), then it is possible to start work with the actual sample.

d) Analysis is done using analytical conditions described in the existing literature.

6. Optimization

During optimization one parameter is changed at a time, and set of conditions are isolated,

rather than using a trial and error approach. Work has been done from an organized

methodical plan, and every step is documented (in a lab notebook) in case of dead ends.

7. Documentation of analytical figures of merit

The originally determined analytical figures of merit limit of quantitation (LOQ), Limit of

detection (LOD), linearity, time per analysis, cost, sample preparation etc., are documented.

8. Evaluation of method development with actual samples

The sample solution should lead to unequivocal, absolute identification of the analyte peak of

interest apart from all other matrix components.

9. Determination of percent recovery of actual sample and demonstration of

quantitative sample analysis:

Percent recovery of spiked, authentic standard analyte into a sample matrix that is shown to

contain no analyte is determined.

Reproducibility of recovery (average +/- standard deviation) from sample to sample and

whether recovery has been optimized has been shown. It is not necessary to obtain 100%

recovery as long as the results are reproducible and known with a high degree of certainty.

The validity of analytical method can be verified only by laboratory studies. Therefore

documentation of the successful completion of such studies is a basic requirement for

determining whether a method is suitable for its intended applications.



Figure 1: Steps in HPLC method development


1. Information on sample, define separation goal

1. Information on sample, define separation goal

2. Need for special HPLC procedure, sample pretreatment, etc.

2. Need for special HPLC procedure, sample pretreatment, etc.

3. Choose proper detection condition

3. Choose proper detection condition

5. Optimize separation condition5. Optimize separation condition

4. HPLC method including preliminary run; estimate best separation condition

4. HPLC method including preliminary run; estimate best separation condition

6. Check for problem or requirement for special problem

6. Check for problem or requirement for special problem

7a. Recover purify material7a. Recover purify material

7b. Quantitative calibration7b. Quantitative calibration

7c. Qualitative method7c. Qualitative method

8. Validate method for release to routine laboratory

8. Validate method for release to routine laboratory


1.3 ANALYTICAL METHOD VALIDATION.

The purpose of the validation is to provide some guidance and recommendation on how to

consider the various validation characteristics for each analytical procedure. Validation of

method is the process of proving that the analytical method is acceptable for its intended

purpose. The validation of analytical method is to be done according to ICH guidelines13,14.

Table 2: ICH validation characteristics versus type of analytical procedure

Analytical Procedure Identification

Impurity testing

Assay

QuantitativeLimit

test

Specificity Yes Yes Yes Yes

Linearity No Yes No Yes

Range No Yes No Yes

Precision

Repeatability No Yes No Yes

Intermediate

PrecisionNo Yes No Yes

Accuracy No Yes No Yes

LOD No No Yes No

LOQ No Yes No No

Robustness No Yes No Yes

Solution Stability Yes Yes Yes Yes



Table 3: Validation Parameters (as per ICH guidelines Q2A)

Parameter Acceptance criteria

Specificity / Selectivity Interference<0.5%

Linearity Correlation Coefficient (r) 0.999

Range

80 – 120 % of target concentration which can be

detected accurately (100±2%) & precisely

(R.S.D. ≤2%)

Precision

Repeatability

R.S.D < 2%*Intermediate

Precision

Accuracy Recovery (98-102%)*. Spike with 80,100,120%.

Detection Limit S/N > 2 or 3

Quantitation Limit S/N >10,RSD<20%*

Robustness

Conditions for HPLC to be varied as follows:

Change in flow rate by ±10%, Change in minor

component of Mobile phase by ±2%, Change in

column oven temperature by ±5°C, Change in

column lot, Change in batch of dosage form.

* Desirable Limit

1.4 SYSTEM SUITABILITY TEST



Prior to the analysis of samples each day, the operator must establish that the HPLC system

and procedures are capable of providing data of acceptable quality. This is accomplished with

system suitability experiments, which can be defined as tests to ensure that the method can

generate results of acceptable accuracy and precision. System suitability parameters for

HPLC are theoretical plate, HETP, retention time, resolution, theoretical plate, tailing factor

etc.

Table 4: System Suitability Parameters and Recommendations

Parameters Recommendations

Capacity Factor (k’) The peak should be well-resolved from

other peaks and the void volume,

generally k’>2.0

Repeatability RSD </= 1% for N >/= 5 is desirable.

Resolution (Rs) Rs of > 1.5 between the peak of interest

and the closest eluting potential

interferent (impurity, excipient,

degradation product, internal standard,

etc.

Tailing Factor (TF or T) T of </= 1.5

Asymmetric Factor (ASM or As) </=2.0

Theoretical Plates (N) In general should be > 2000



1.5 Drug Profile

Albendazole

Table 5: Physiochemical Properties

CAS number

53965-21-8

CBNumber

CB0241320

Chemical structure

Chemical name Albendazole

Molecular formula C12H15N3O2S

Molecular wt(g/mol) 265.33g/mol

Appearance Blue color liquid suspension

Melting Point 208-210°C

Solubility Soluble in acidified methnol

Table 6: Pharmacological properties

Therapeutic category Anthelmintic

Mechanism of action Albendazole has larvicidal effects in

necatoriasis and ovicidal effects in ascariasis,

ancylostomiasis, and trichuriasis by

degenerative alterations in the tegument and

intestinal cells of the worm by binding to the

colchicine-sensitive site of tubulin, thus

inhibiting its polymerization or assembly into

microtubules.

Indications Hepatic functions have to be obtained regularly


http://en.wikipedia.org/wiki/Tubulin

http://en.wikipedia.org/wiki/Colchicine

http://en.wikipedia.org/wiki/Tegument

http://en.wikipedia.org/wiki/Trichuriasis

http://en.wikipedia.org/wiki/Ancylostomiasis

http://en.wikipedia.org/wiki/Ascariasis

http://en.wikipedia.org/wiki/Necatoriasis


in patients receiving albendazole

Bioavailability 20-50%

Half life 8.5 hr.

2. LITERATURE REVIEW

2.1 OFFICAL METHODS

2.1.1 ALBENDAZOLE ORAL SUSPENSION

Albendazole is official in USP 31

2.2.1 OTHER PUBLISHED METHODS

Rajshekhar V et.al.(1997 ) did Validation of diagnostic criteria for solitary cerebral cysticercus granuloma in patients presenting with seizures. Objective was to evaluate a set of clinical and computed tomographic (CT) criteria (previously described by us) to predict the diagnosis of a solitary cerebral cysticercus granuloma (SCCG) at initial presentation, in patients presenting with seizures.

Ragno G et. al. (2006) did Photo- and thermal-stability studies on benzimidazole anthelmintics by HPLC and GC-MS. Photo- and thermal-stability of the anthelmintics Albendazole, Mebendazole and Fenbendazole as in solid as in solution form has been investigated, by using a Xenon arc lamp as a radiation source, according to the ICH guideline for the drug stability tests. The degradation process was monitored by a HPLC method. All drugs showed high photosensitivity in solution but a reliable stability in solid form and when exposed to a temperature up to 50 degrees C. Two main degradation products from hydrolysis of the carbamic groups were identified by GC-MS. Validation studies demonstrated high accuracy (recovery 94 to 106%) and precision (RSD under 4.6%) of the HPLC method. The analytical procedure was successfully applied to the control of the drugs in the respective pharmaceutical formulations.

Nozal MJ et. al. (2002) did Separation of albendazole sulfoxide enantiomers by chiral supercritical-fluid chromatography. The enantioseparation of albendazole sulfoxide (ABZSO) by chiral supercritical-fluid chromatography


http://www.ncbi.nlm.nih.gov/pubmed/12543509


http://www.ncbi.nlm.nih.gov/pubmed?term=%22del%20Nozal%20MJ%22%5BAuthor%5D



http://www.ncbi.nlm.nih.gov/pubmed?term=%22Ragno%20G%22%5BAuthor%5D



http://www.ncbi.nlm.nih.gov/pubmed?term=%22Rajshekhar%20V%22%5BAuthor%5D


(SFC) on two columns, based on the polysaccharide derivatives Chiralpak AD and Chiralcel OD, was studied. The effect of different modifiers, methanol, ethanol, 2-propanol, and acetonitrile, was examined. The results showed that ABZSO can be separated on both columns, using an alcohol-type modifier. Using the Chiralpak AD column, the best results were obtained with 2-propanol and, in the case of the Chiralcel OD, with methanol.

Nobilis M et. al. (2007) did Achiral and chiral high-performance liquid chromatographic determination of flubendazole and its metabolites in biomatrices using UV photodiode-array and mass spectrometric detection.Flubendazole, methyl ester of [5-(4-fluorobenzoyl)-1H-benzimidazol-2-yl]carbamic acid, belongs to the group of benzimidazole anthelmintics, which are widely used in veterinary and human medicine. The phase I flubendazole biotransformation includes a hydrolysis of the carbamoyl methyl moiety accompanied by a decarboxylation (hydrolysed flubendazole) and a carbonyl reduction of flubendazole (reduced flubendazole). Flubendazole is a prochiral drug, hence a racemic mixture is formed during non-stereoselective reductions at the carbonyl group. Two bioanalytical HPLC methods were developed and validated for the determination of flubendazole and its metabolites in pig and pheasant hepatic microsomal and cytosolic fractions. Analytes were extracted from biomatrices into tert-butylmethyl ether. The first, achiral method employed a 250 mm x 4 mm column with octylsilyl silica gel (5 microm) and an isocratic mobile phase acetonitrile-0.025 M KH(2)PO(4) buffer pH 3 (28:72, v/v). Albendazole was used as an internal standard. The whole analysis lasted 27 min at a flow rate of 1 ml/min. The second, chiral HPLC method, was performed on a Chiralcel OD-R 250 mm x 4.6 mm column with a mobile phase acetonitrile-1 M NaClO(4) (4:6, v/v). This method enabled the separation of both reduced flubendazole enantiomers. The enantiomer excess was evaluated. The column effluent was monitored using a photodiode-array detector (scan or single wavelength at lambda=246 nm). Each of the analytes under study had characteristic UV spectrum, in addition, their chemical structures were confirmed by high-performance liquid chromatography-mass spectrometry (HPLC-MS) experiments. Stereospecificity in the enzymatic carbonyl reduction of flubendazole was observed. While synthetic racemic mixture of reduced flubendazole was separated to equimolar amounts of both enantiomers, practically only one enantiomer was detected in the extracts from all incubates.

Ruyck H et.al. (2002) did Development and validation of a liquid chromatographic-electrospray tandem mass spectrometric multiresidue method for anthelmintics in milk.A liquid chromatographic-tandem mass spectrometric multiresidue method for the simultaneous quantitative determination of the tetrahydroimidazole, levamisole and the benzimidazoles





http://www.ncbi.nlm.nih.gov/pubmed?term=%22De%20Ruyck%20H%22%5BAuthor%5D




http://www.ncbi.nlm.nih.gov/pubmed?term=%22Nobilis%20M%22%5BAuthor%5D


thiabendazole, oxfendazole, oxibendazole, albendazole, fenbendazole, febantel and triclabendazole in milk has been developed and validated. The anthelmintic residues were extracted with ethyl acetate. The liquid chromatographic separation was performed on a reversed-phase C18 column with gradient elution. The analytes were detected by tandem quadrupole mass spectrometry after positive electrospray ionisation by multiple reaction monitoring. The confirmatory method is very sensitive and each component can be detected at a residue level lower than 1 microgram/l. The method is validated according to the revised European Union requirements and all parameters were found conform the criteria. The evaluated parameters were linearity, specificity, stability, recovery, precision (repeatability and within-laboratory reproducibility) and analytical limits (detection limit, decision limit and detection capability). This analytical method is applied in the Belgian monitoring programme for classical anthelmintic veterinary drugs in raw farm cow's milk.

Nobilis M et. al. (2008) did Sensitive chiral high-performance liquid chromatographic determination of anthelmintic flubendazole and its phase I metabolites in blood plasma using UV photodiode-array and fluorescence detection Application to pharmacokinetic studies in sheep. Although benzimidazole anthelmintic flubendazole, methyl ester of [5-(4-fluorobenzoyl)-1H-benzimidazol-2-yl]carbamic acid, is extensively used in veterinary and human medicine for the treatment of gastrointestinal parasitic helminth infections, reliable data about its pharmacokinetics in various species have not been reported. Our previous work [M. Nobilis, Th. Jira, M. Lísa, M. Holcapek, B. Szotáková, J. Lamka, L.Skálová, J. Chromatogr. A 1149 (2007) 112-120] had described the stereospecificity of carbonyl reduction during phase I metabolic experiments in vitro. For in vivo pharmacokinetic studies, further improvement and optimization of bioanalytical HPLC method in terms of sensitivity and selectivity was necessary. Hence, a modified chiral bioanalytical HPLC method involving both UV photodiode-array and fluorescence detection for the determination of flubendazole, both enantiomers of reduced flubendazole and hydrolyzed flubendazole in the extracts from plasma samples was tested and validated. Albendazole was used as an internal standard. Sample preparation process involved a pH-dependent extraction of the analytes from the blood plasma into tert-butylmethyl ether. Chromatographic separations were performed on a Chiralcel OD-R 250 mm x 4.6mm column with mobile phase methanol-1M NaClO(4) (75:25, v/v) at the flow rate 0.5 ml min(-1). In quantitation, selective UV absorption maxima of 290 nm (for reduced flubendazole), 295 nm (for albendazole), 310 nm (for flubendazole) and 330 nm (for hydrolyzed flubendazole) were used in the UV photodiode-array detection, and lambda(exc.)/lambda(emis.)=228 nm/310 nm (for reduced flubendazole) and lambda(exc.)/lambda(emis.)=236 nm/346 nm (for albendazole) were set on the fluorescence detector. The fluorescence





http://www.ncbi.nlm.nih.gov/pubmed?term=%22Nobilis%20M%22%5BAuthor%5D


detection was approximately 10-times more sensitive than the UV detection. Each HPLC run lasted 27 min. The validated chiral HPLC-PDA-FL method was employed in the pharmacokinetic studies of flubendazole in sheep. The stereospecificity of the enzymatic carbonyl reduction of flubendazole was also observed in vivo. (+)-Reduced flubendazole was found to be the principal metabolite in ovine blood plasma and only low concentrations of hydrolyzed flubendazole, the parent flubendazole and (-)-reduced flubendazole were detected in this biomatrix.

Bonato PS et. al. (2007) did Simultaneous determination of albendazole metabolites, praziquantel and its metabolite in plasma by high-performance liquid chromatography-electrospray mass spectrometry. The analysis of albendazole sulfoxide, albendazole sulfone, praziquantel and trans-4-hydroxypraziquantel in plasma was carried out by high-performance liquid chromatography-mass spectrometry ((LC-MS-MS). The plasma samples were prepared by liquid-liquid extraction using dichloromethane as extracting solvent. The partial HPLC resolution of drug and metabolites was obtained using a cyanopropyl column and a mobile phase consisting of methanol:water (3:7, v/v) plus 0.5% of acetic acid, at a flow rate of 1.0 mL/min. Multi reaction monitoring detection was performed by electrospray ionization in the positive ion mode, conferring additional selectivity to the method. Method validation showed relative standard deviation (precision) and relative errors (accuracy) lower than 15% for all analytes evaluated. The quantification limit was 5 ng/mL and the linear range was 5-2500 ng/mL for all analytes. The method was used for the determination of drug and metabolites in swine plasma samples and proved to be suitable for pharmacokinetic studies.

Batzias GC et. al. (2004) did Quantitative determination of albendazole metabolites in sheep spermatozoa and seminal plasma by liquid chromatographic analysis with fluorescence detection. A new analytical method for the simultaneous quantitative determination of albendazole metabolites in sheep spermatozoa and seminal plasma at levels down to 46.5 ng/mL for albendazole sulphoxide (ABZ-SO), 7.5 ng/mL for albendazole sulphone (ABZ-SO2) and 12 ng/mL for albendazole 2-aminosulphone (ABZ-SO2NH2) has been developed. Analytes were extracted from alkalinized samples with ethyl acetate. Separation was carried out on a C18 column in the presence of tetra-n-butylammonium (TBA) hydrogen sulphate and octanesulphonate sodium (OCT), as ion-pair agents. Fluorometric detection was performed with excitation and emission wavelengths set at 290 and 320 nm, respectively. Accuracy data showed overall recoveries (+/-S.E.M.) of 83.1+/-1.2% for ABZ-SO, 98.8+/-0.6% for ABZ-SO2 and 85.3+/-0.7% for ABZ-SO2NH2, in spermatozoa. Respective values in seminal plasma were 88.0+/-0.9%, 97.7+/-0.5% and 93.2+/-1.7%. Precision data suggested coefficient of variation (CV%) values lower than 5.9% for spermatozoa and





http://www.ncbi.nlm.nih.gov/pubmed?term=%22Batzias%20GC%22%5BAuthor%5D




http://www.ncbi.nlm.nih.gov/pubmed?term=%22Bonato%20PS%22%5BAuthor%5D


3.8% for seminal plasma. The method was successfully applied for the determination of the three albendazole metabolites in semen samples collected from rams that had been orally administered albendazole.

Chen X et. al, (2004) did Simultaneous determination of albendazole and its major active metabolite in human plasma using a sensitive and specific liquid chromatographic-tandem mass spectrometric method. A method for the simultaneous determination of albendazole (ABZ) and its major active metabolite albendazole sulfoxide (ABZ-SO) was developed and validated. The analytes were extracted from plasma samples by liquid-liquid extraction and analyzed using liquid chromatography-tandem mass spectrometry with an electrospray ionization interface. Estazolam was used as the internal standard. The assay was linear in the concentration range 0.4-200 ng/ml for ABZ and 4.0-2000 ng/ml for ABZ-SO. The intra- and inter-run precision (R.S.D.), calculated from quality control (QC) samples was less than 7.1 and 9.4% for ABZ and ABZ-SO, respectively. The accuracy as determined from QC samples was within +/- 3% for the analytes. Recoveries of ABZ and ABZ-SO were greater than 77 and 53%, respectively, over the calibration curve range. The method developed was successfully applied to pharmacokinetic studies of ABZ and ABZ-SO after an oral dose of 400 mg albendazole to healthy volunteers.





http://www.ncbi.nlm.nih.gov/pubmed?term=%22Chen%20X%22%5BAuthor%5D


3. OBJECTIVES AND METHODOLOGY

WORK TO BE PERFORMED IV SEMESTER

Development of analytical techniques for Alendazole in oral Suspension dosage form.

Validation of Developed methods as per ICH guidelines (specificity, selectivity, linearity,

range, precision, accuracy, limit of quantitation, and limit of detection)

Application of the Developed Method for Albendazole Oral Suspension

Physical and Chemical characterization of Albendazole

Determination of absorbance maxima of Albendazole

Determination of suitable solvent for sample preparation

Mobile phase selection

Organic solvent: Acetonitrile, Methanol

Aqueous solvent: Water

Selection of suitable PH

Selection of column & column temperature

Optimization of mobile phase, column & solvent system

4 PLAN OF WORK

METHOD DEVELOPMENT AND VALIDATION OF ESTIMATION OF ASSAY FOR ALBENDAZOLE

ORAL SUSPENSION



Literature survey on- I. Drug and analytical method to be used.

II. Reviewing of estimation methods.

Method development and optimization for the estimation of Blonanserin considering variables such as mobile phase ratio, pH, gradient, flow rate, temperature, solvent type and the column and its type. Forced degradation studies

Validation of the method on establishment of specificity/selectivity followed by other parameters like accuracy, precision, linearity, range, robustness, lod and loq.

Compilation of data & preparation for publication

Preparation of report and thesis

6. REFERENCES

1.The Aaps journal 2007; 9(1) article 5.



2. Journal of veterinary and pharmacology, volume 28, issue 4, page 377-384, august 2005.

3. http://www.medicinenet.com/albendazole/articl.

4.Wikipedia of Albendazole.

5.Lanusse CE, Gascon LR, Prichard RK: Comparative plasma disposition kinetics of albendazole, fenbendazole, oxfenbendazole and their metabolites in adult sheep. J Vet Pharmacol Therap 1995;18:196-203.

6. Lifschitz A, Virkel G, Mastromarino M, Lanusse C: Enhanced plasma availability of the metabolites of albendazole in fasted adult sheep. Vet Res Commun1997;21:201-211.

7. Evrard B, Chiap P, DeTullio P, et al: Oral bioavailability in sheep of albendazole from a suspension and from a solution containing hydroxypropyl-beta-cyclodextrin.J Control Release 2002;85:45-50.

8. Marriner SE, Bogan JA: Pharmacokinetics of albendazole in sheep. Am J Vet Res 1980;41:1126-1129.

9. Alvarez LI, Sanchez SF, Lanusse CE : In vivo and ex-vivo uptake of albendazole and its sulphoxide metabolite by cestode parasites: relationship with their kinetic behaviourin sheep. J Vet Pharmacol Ther 1999;22:77-86.

10. Lifschitz A, Pis A, Alvarez L, et al: Bioequivalence of ivermectin formulations in pigs and cattle. J Vet Pharmacol Ther 1999;22:27-34.

11. US Food and Drug Administration: Bioequivalence guidance. Rockville MD: Center for Veterinary Medicine (CVM), Food and Drug Administration;2002:1-28.

12. USP 25–NF 20, Validation of Compendial Methods Section (1225) (United States Pharmacopeal Convention, Rockville, Maryland, USA, 2002) p 2256.

13. G.A. Shabir, "Validation of HPLC Chromatography Methods for Pharmaceutical Analysis. Understanding the Differences and Similarities Between Validation Requirements of FDA, the US Pharmacopeia and the ICH," J. Chromatogr. A. 987(1-2), 57-66 (2003).

14. C.E. Wood, "Medicare Program; Changes to the Hospital Outpatient Prospective," Med. J. Aust. 165, 510–514 (1996).

15. A. Prentice, "Medical Management of Menorrhagia," Br. Med. J. 319, 1343–1345 (1999).

16. D.T. Baired and A.F. Glasier, "Hormonal Contraception," New Engl. J. Med. 328, 1543–1549 (1993).

17. P.E. Belchetz, "Hormonal Treatment of Postmenopausal Women," New Engl. J. Med. 330, 1062–1071(1994).




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