“DEVELOPMENT OF NEW ANALYTICAL METHOD AND ITS VALIDATION FOR THE DETERMINATION OF MELOXICAM AND CHLORAMPHENICOL IN BULK AND MARKETED FORMULATIONS”
MASTER OF PHARMACY DISSERTATION PROTOCOL
SUBMITTED TO THE
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA, BANGALORE
BY
BAROCHIA KAUSHAL GAUTAMBHAI
Under The Guidance of
Dr. E.V.S. Subrahmanyam. M.PHARM. Ph.D
P.G. DEPARTMENT OF QUALITY ASSURANCE,
SRINIVAS COLLEGE OF PHARMACY, MANGALORE – 574143
2010 – 2011
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
BANGALORE, KARNATAKA
ANNEXURE – ІІ
REGISTRATION OF SUBJECT FOR DISSERTATION
1.0 / NAME OF THE CANDIDATEADDRESS / BAROCHIA KAUSHAL GAUTAMBHAI.
DEPARTMENT OF Q.A.,
SRINIVAS COLLEGE OF PHARMACY,
VALACHIL,POST PARENGIPITTE,
MANGALORE TQ-574143
2.0 / NAME OF THE INSTITUTION / SRINIVAS COLLEGE OF PHARMACY, VALACHIL, MANGALORE.
3.0 / COURSE OF STUDY &
SUBJECT / MASTER OF PHARMACY
(QUALITY ASSURANCE)
4.0 / DATE OF ADMISSION / 31st MAY 2010
5.0 / TITLE OF THE TOPIC:
“DEVELOPMENT OF NEW ANALYTICAL METHOD AND ITS VALIDATION FOR THE DETERMINATION OF MELOXICAM AND CHLORAMPHENICOL IN BULK AND MARKETED FORMULATIONS”
6.0
6.1
6.2
6.3
6.4
7.0
7.1
7.2
8.0 / BRIEF RESUME OF THE INTENDED WORK:
NEED FOR STUDY:
Analytical Method Development for Pharmaceutical Formulations:
Analytical methods are essential to characterize drug substances and drug products composition during all stages of pharmaceutical development. For routine analytical purpose it is always necessary to establish methods capable of analyzing huge number of samples in a short time period with high accuracy and precisionThe number of drugs, which may be either new entities or partial structural modification of the existing ones, introduced into the market is increasing every year. Very often there is a time lag from the date of introduction of a drug into the market to the date of its inclusion in pharmacopoeias. Hence, standards and analytical procedures for these drugs may not be available in the pharmacopoeias. It becomes necessary, therefore to develop new analytical methods for such drugs. These products can present challenges to the analytical chemist responsible for the development andvalidation of analytical methods.
Basic criteria for new method development of drug analysis:
· The drug or drug combination may not be official in any pharmacopoeias.· A proper analytical procedure for the drug may not be available in the literature due to patent regulations.
· Analytical methods may not be available for the drug in the form of a formulation due to the interference caused by the formulation excipients.
· Analytical methods for a drug in combination with other drugs may not be available.
· The existing analytical procedures may require expensive reagents and solvents. It may also involve cumbersome extraction and separation procedures and these may not be reliable.
Analytical method development provides the support to track the quality of the product from batch to batch. Estimation can be performed by the following two methods:
· Titrimetric methods and
· Instrumental methods.
§ Spectrophotometric Methods
§ Chromatographic Methods
Methods for analyzing drugs in dosage forms can be developed, provided one has knowledge about the nature of the sample, its molecular weight, polarity, ionic character and the solubility parameter. Method development involves considerable trial and error procedures. The most difficult problem usually is where to start, what type of column is worth trying with what kind of mobile phase.
The following is a suggested method development scheme for a typical HPLC-UV
related substance method.
1. To define the goals for method development (e.g., what is the intended use of the method?), and to understand the chemistry of the analytes and the drug product.
2. To develop preliminary HPLC conditions to achieve minimally acceptable separations. These HPLC conditions will be used for all subsequent method development experiments.
3. To develop a suitable sample preparation scheme for the drug product
4. To determine the appropriate standardization method and the use of relative response factors in calculations.
5. To identify the “weaknesses” of the method and optimize the method through experimental design. Understand the method performance with different conditions, different instrument set ups and different samples.
6. To complete method validation according to ICH guidelines as mentioned in
Q2 (R1).
A BRIEF INTRODUCTION ABOUT MELOXICAM 1
MELOXICAM:
IUPAC NAME: 4-hydroxy-2-methyl-N-(5-methyl-2-thiazolyl)-2H-1,2-benzothiazine-3-carboxamide-1,1-dioxide.
MOLECULAR FORMULA: C14H13N3O4S2
MOLECULAR WEIGHT: 351.403
CATEGORY: NON-STEROIDAL ANTI- INFLAMMATORY DRUG
BIOAVAILABILITY: 89%
PROTEIN BINDING : 99.4%
METABOLISM: Hepatic
HALF-LIFE: 15 to 20 hours
EXCRETION: Urine and faeces equally
PHARMACOLOGY:
MECHANISM OF ACTION:
Meloxicam inhibits cyclooxygenase (COX), the enzyme responsible for converting arachidonic acid into prostaglandin H2—the first step in the synthesis of prostaglandins, which are mediators of inflammation. Meloxicam has been shown, especially at its low therapeutic dose, selectively to inhibit COX-2 over COX-1.
Meloxicam concentrations in synovial fluid range from 40% to 50% of those in plasma. The free fraction in synovial fluid is 2.5 times higher than in plasma, due to the lower albumin content in synovial fluid as compared to plasma. The significance of this penetration is unknown, but it may account for the fact that it performs exceptionally well in treatment of arthritis in animal models.
ADVERSE EFFECT:
Meloxicam use can result in gastrointestinal toxicity and bleeding, tinnitus, blinding headaches, rash, very dark or black stool (sign of intestinal bleeding). It has fewer gastrointestinal side effects than diclofenac, piroxicam, naproxen, and perhaps all other NSAIDs which are not COX-2 selective.[4] Although meloxicam does inhibit thromboxane A, it does not appear to do so at levels that would interfere with platelet function.
REVIEW OF LITERATURE:
A literature survey was carried out for the estimation of meloxicam in bulk and marketed dosage forms. It was found that a very few methods have been reported for this drug. The collection of references are reproduced below:
· Emirhan nemuthi / Sedef kir2 have developed validated method to determine meloxicam Tablet by using UV-spectrophotometry .for determination of meloxicam they used 100 mM boret buffer ( pH 8.5 ).the spectrum was recorded from 200nm to 450nm. The quantitative analysis is carried out at 363nm.This applied in six preparation including two formulation.The data from developed method was statically compared with capillary electrophoretic method in literature.In this method they used megnesium hydroxide , sodium hydroxide , and boric acid throughout experiment.
· Farzana SB , Pradeep RV 3 have developed RP-HPLC method for Determination of meloxicam in bulk and commercial preparation .RP-HPLC was developed by using C18 HI Q SIL column with acetonitrile –water-glacial acetic acid [55:40:5(%v/v)] at a flow rate 1ml/minute and detection at wavelength of 355nm.As a result a sharp peak was obtained for MLX at a rate of 6.8±0.01 min.
· Haixia Z and Hoo-kyun C4 have developed method for analysis of meloxicam by HPLC with cloud point extraction in human serum . for this method they extract meloxicam from serum sample after adding 1 ml of 3%v/v triton -114 aquous solution in presence of 1M HCL and 60 mg Nacl .It is developed in C18 analytical column with a mobile phase aquous acetic acid 1%v/v and acitonitrile (54:46v/v).UV detection performed at 360 nm.
· Khan F, Lohiya RT,Umerkar M,5 have been developed UV spectrophotometry method for simultaneous estimation of meloxicam and paracetamol in tablet by simultanious equation , absorbance ratio and absorbance correction method.
For method 1-wave length selected were 257.6 for meloxicam and 270.6nm for paracetamol.
For method 2-257.6nm for paracetamol and 297.6 nm isoabsorptive point for paracetamol and meloxicam
For method 3-362 nm for correction method
Solution used throughout experiment is 0.1N NaOH and whatmann filter paper.
§ Jung woo B, Mi-jeong K,Choon-gon J and Seok-yong L6 had developed validated method of meloxicam using HPLC with UV detection . In experiment they use SUNFIREC 18 column (150nm×4.6nm,5µm) with mobile phase of acetonitrile 20mM ,potassium hydrogen phosphate (40:60v/v, pH 3.5).UV detection is carried out at wavelength 355nm.The flow rate was 1.2 ml/minute.
· Hassan EM.7 has developed Spectrophotometric and fluorimetric methods for the determination of meloxicam in dosage forms. Four simple and accurate methods were presented for the determination of meloxicam in dosage forms. These methods were based on: the direct measurements of the differential spectra at 339.9-384.7 nm (A), the 1D-values at 322-368 nm and 2D-values at 343.2-385.6 nm (B), the formation of an ion-association complex between the drug and safranin T with subsequent absorption measurement at 518 nm (C) and fluorescence measurement at 582 nm (D). All variables were studied to optimize the formation of the ion-association complex. Beer's law was valid over the concentration range 2-10 microg ml(-1) (method A), 1-10 µg. ml(-1) (method B), 4.0-12 µg ml(-1) (method C) and 0.4-1.2 microg ml(-1) (method D). The detection limits were 0.11, 0.07, 0.10, 0.33 and 8.74 x 10(-3) microg ml(-1) for methods A, B, C and D, respectively. The proposed methods were successfully applied to the assay of meloxicam in tablets and suppositories. .
A BRIEF INTRODUCTION OF CHLORAMPHENICOL8
IUPAC NAME:2,2-dichloro-N-[(1R,2R)-2-hydroxy-1-(hydroxymethyl)-2-(4-nitrophenyl)ethyl] acetamide
FORMULA: C11H12Cl2N2O5
MOLECULAR WEIGHT: 323.132
BIOAVAILABILITY: 75-90%
METABOLISM: HEPATIC
HALF LIFE: 1.5-4.0 HOURS
EXCRETION: RENAL
ROUTES:TOPICAL(OCULAR),ORAL,INTRAVENOUS,INTRAMUSCULAR
Spectrum of activity
Because it functions by inhibiting bacterial protein synthesis, chloramphenicol has a very broad spectrum of activity: it is active against Gram-positive bacteria (including most strains of MRSA), Gram-negative bacteria and anaerobes. It is not active against Pseudomonas aeruginosa, Chlamydiae, or Enterobacter species. It has some activity against Burkholderia pseudomallei, but is no longer routinely used to treat infections caused by this organism (it has been superseded by ceftazidime and meropenem). In the West, chloramphenicol is mostly restricted to topical uses because of the worries about the risk of aplastic anaemia
Adverse effect:
Aplastic anemia ,Bone marrow suppression, Leukemia, tinnitus, Gray baby syndrome ,
Therapeutic uses:
The original indication of chloramphenicol was in the treatment of typhoid, but the now almost universal presence of multi-drug resistant Salmonella typhi has meant that it is seldom used for this indication except when the organism is known to be sensitive. Chloramphenicol may be used as a second-line agent in the treatment of tetracycline-resistant cholera.
Spectrum of activity
Because it functions by inhibiting bacterial protein synthesis, chloramphenicol has a very broad spectrum of activity: it is active against Gram-positive bacteria (including most strains of MRSA), Gram-negative bacteria and anaerobes. It is not active against Pseudomonas aeruginosa, Chlamydiae, or Enterobacter species. It has some activity against Burkholderia pseudomallei, but is no longer routinely used to treat infections caused by this organism (it has been superseded by ceftazidime and meropenem). In the West, chloramphenicol is mostly restricted to topical uses because of the worries about the risk of aplastic anaemia.
Pharmacokinetics
Chloramphenicol is extremely lipid soluble, it remains relatively unbound to protein and is a small molecule: it has a large apparent volume of distribution of 100 litres and penetrates effectively into all tissues of the body, including the brain. The concentration achieved in brain and cerebrospinal fluid (CSF) is around 30 to 50% even when the meninges are not inflamed; this increases to as high as 89% when the meninges are inflamed. Chloramphenicol increases the absorption of iron.
Drug interactions
Administration of chloramphenicol concomitantly with bone marrow depressant drugs is contraindicated, although concerns over aplastic anaemia associated with ocular chloramphenicol have largely been discounted.
Chloramphenicol is a potent inhibitor of the cytochrome P450 isoforms CYP2C19 and CYP3A4 in the liver. Inhibition of CYP2C19 causes decreased metabolism and therefore increased levels of, for example, antidepressants, antiepileptics and proton pump inhibitors if they are given concomitantly. Inhibition of CYP3A4 causes increased levels of, for example, calcium channel blockers, immunosuppressants, chemotherapeutic drugs, benzodiazepines, azole antifungals, tricyclic antidepressants, macrolide antibiotics, SSRIs, statins and PDE5 inhibitors.
Mechanism of action
Chloramphenicol is bacteriostatic (that is, it stops bacterial growth). It is a protein synthesis inhibitor, inhibiting peptidyl transferase activity of the bacterial ribosome, binding to A2451 and A2452 residues in the 23S rRNA of the 50S ribosomal subunit, preventing peptide bond formation. While chloramphenicol and the macrolide class of antibiotics both interact with ribosomes, chloramphenicol is not a macrolide. Chloramphenicol directly interferes with substrate binding; macrolides sterically block the progression of the growing peptide.
Resistance
There are three mechanisms of resistance to chloramphenicol: reduced membrane permeability, mutation of the 50S ribosomal subunit and elaboration of chloramphenicol acetyltransferase. It is easy to select for reduced membrane permeability to chloramphenicol in vitro by serial passage of bacteria, and this is the most common mechanism of low-level chloramphenicol resistance. High-level resistance is conferred by the cat-gene; this gene codes for an enzyme called chloramphenicol acetyltransferase which inactivates chloramphenicol by covalently linking one or two acetyl groups, derived from acetyl-S-coenzyme A, to the hydroxyl groups on the chloramphenicol molecule. The acetylation prevents chloramphenicol from binding to the ribosome. Resistance-conferring mutations of the 50S ribosomal subunit are rare.
REVIEW OF LITERATURE:
Ø Charles JL, Noreen JW, George FJ, and Harvey MS9 have described quantitative Gas-Chromatographic Flame-ionization method for chloramphenicol in human serum. The serum (500 l) was extracted into ethyl acetate and nonpolar impurities were subsequently partitioned into hexane. The drug is chromatographed as its bis-trimethylsilyl derivative, with the analog thiamphenicol as the internal standard. Within-run precision (CV) is 4.4% at a serum concentration of 41 mg/liter and 9.2% at a concentration of 5 mg/liter. Over a six-month period, the run-to-run variation was 5.1 % at 40 mg/liter (n = 24). Results by the gas-chromatographic method compared well with those by an established colorimetric procedure; mean concentrations for the comparison samples in the two procedures were 18.4 mg/liter and 17.6 mg/liter, respectively (n = 27), with a coefficient of correlation of 0.998. The gas-chromatographic method is more precise and specific than classical microbiological procedures and is suitable for routine therapeutic monitoring.
Ø Gasilova NV, Eramine SA10 have developed method for determination of chloramphenicol in milk by a fluorescence polarization immunoassay. The optimum pairs of antibodies and antigens labeled with fluorescein were chosen, and the analytical characteristics of the procedure were determined. A rapid procedure for milk sample preparation with the use of a saturated solution of ammonium sulfate was optimized. The total time of sample preparation and determination of chloramphenicol in milk was no longer than 10 min. The detection limits of chloramphenicol in water and milk were 10ng/mL and 20μg/kg, respectively. The procedure developed for the determination of chloramphenicol was tested in the analysis of model and real milk samples. It was found that some milk samples contained chloramphenicol in concentrations of 38–41 μg/kg, which are several times higher than the maximum permissible concentration (MPC) (10 μg/kg).