Crohn's disease is a type of inflammatory bowel disease (IBD) that may affect any part of the gastrointestinal (GI) tract, from mouth to anus

Crohns disease

Crohn's disease is a type of inflammatory bowel disease (IBD) that may affect any part of the gastrointestinal (GI) tract, from mouth to anus.[1] It was named after gastroenterologist Burrill Bernard Crohn, in 1932, along with colleagues Leon Ginzburg and Gordon Oppenheimer at Mount Sinai Hospital in New York, described a series of patients with inflammation of the terminal ileum of the small intestine, the area most commonly affected by the illness.[2]While symptoms vary from patient to patient and some may be more common than others, the tell-tale symptoms of Crohns disease include symptoms related to inflammation of the GI tract and general symptoms that may also be associated with IBD.[1] Symptoms often include: abdominal cramps and pain, persistent diarrhea (which may be bloody if inflammation is severe), urgent need to move bowels, sensation of incomplete evacuation and constipation (which can lead to bowel obstruction). General symptoms include fever, weight loss, loss of appetite, fatigue, night sweats and loss of normal menstrual cycle.[3]Crohn's disease is caused by a combination of environmental, immune and bacterial factors in genetically susceptible individuals.[4],[5],[6] It results in a chronic inflammatory disorder, in which the body's immune system attacks the gastrointestinal tract possibly directed at microbial antigens.[7] While Crohn's is an immune related disease, it does not appear to be an autoimmune disease (in that the immune system is not being triggered by the body itself).[8] The exact underlying immune problem is not clear; however, it may be an immunodeficiency state.[9] About half of the overall risk is related to genetics with more than 70 genes found to be involved.[3] Tobacco smokers are two times more likely to develop Crohn's disease than nonsmokers.[10] It also often begins after gastroenteritis. Diagnosis is based on a number of findings including: biopsy and appearance of the bowel wall, medical imaging and description of the disease. Other conditions that can present similarly include irritable bowel syndrome and Behcet's disease.[3]There are no medications or surgical procedures that can cure Crohn's disease. Treatment options help with symptoms, maintain remission, and prevent relapse. In those newly diagnosed a corticosteroid may be used for a brief period of time to quickly improve the disease with another medication such as either methotrexate or a thiopurine used to prevent recurrence. An important part of treatment is the stopping of smoking among those who do. One in five people with the disease are admitted to hospital each year, and half of those with the disease will require surgery for the disease at some point over a ten year period. While surgery should be used as little as possible, it is necessary to address some abscesses, certain bowel obstructions, and cancers. Checking for bowel cancer via colonoscopy is recommended every few years, starting eight years after the disease has begun.[3]Crohn's disease affects about 3.2 per 1,000 people in Europe and North America,[11] but it is less common in Asia and Africa.[12] Crohns has historically been more common in developed rather than undeveloped countries, in urban rather than rural areas, and in northern rather than southern climates.[13] However, rates have been increasing, particularly in the developing world since the 1970s.[14] Inflammatory bowel disease resulted in 35,000 deaths in 2010[15] and those with Crohn's disease have a slightly reduced life expectancy.[3] The disease tends to start in the teens and twenties, although it can occur at any age.[1] Males and females are equally likely to be affected.[1] Crohns tends to run in families, so if a person or a close relative have the disease, the family members have a significantly increased chance of developing Crohns. Studies have shown that 5-20% of affected individuals have a first degree relative (parents, child, or sibling) with one of the diseases. The risk is greater with Crohns disease than ulcerative colitis. The risk is also substantially higher when both parents have IBD.Crohns Disease Medication Options

Management of Crohn's disease involves first treating the acute symptoms of the disease, then maintaining remission. Since Crohn's disease is an immune system disease, it cannot be cured by medication or surgery. Treatment for Crohn's disease usually involves drug therapy or, in certain cases, surgery. There is currently no cure for the disease, and there is no one treatment that works for everyone. Doctors use one of two approaches to treatment either step-up (starts with milder drugs first), or top-down (gives people stronger drugs earlier in the treatment process).

The goal of medical treatment is to reduce the inflammation that triggers the signs and symptoms. It is also to improve long-term prognosis by limiting complications. In the best cases, this may lead not only to symptom relief but also to long-term remission. Successful medical treatment accomplishes two important goals: it allows the intestinal tissue to heal and it also relieves the symptoms of fever, diarrhea, and abdominal pain. Surgery may be required for complications such as obstructions or abscesses, or if the disease does not respond to drugs within a reasonable time. However, surgery cannot cure Crohn's disease by any means - it involves removing the diseased part of the intestine and rejoining the healthy ends, as Crohn's disease tends to recur after surgery. Once the symptoms are brought under control (inducing remission), medical therapy is used to decrease the frequency of disease flares (maintaining remission or maintenance).[16]There are several groups of drugs to treat Crohns disease today. They are:

Anti-inflammatory drugs

Anti-inflammatory drugs are often the first step in the treatment of inflammatory bowel disease. They include Oral 5-aminosalicylates and corticosteroids. Oral 5-Aminosalicylates (5-ASA): these drugs may be helpful if Crohn's disease affects the colon, but they aren't helpful treating disease in the small intestine. They are medications that contain 5-aminosalicylate acid (5-ASA), which helps control inflammation. Health care providers use aminosalicylates to treat people newly diagnosed with Crohn's disease who have mild symptoms. Aminosalicylates include Sulfasalazine (Azulfidine) which contains sulfa, Mesalamine (Asacol, Delzicol, Pentasa, Lialda, Apriso), Olsalazine and Balsalazide.These drugs are not specially approved by the Food and Drug Administration (FDA) for use in Crohns. Especially sulfasalazine, have a number of side effects, including nausea, diarrhea, vomiting, heartburn and headache. However, they can work at the level of the lining of the GI tract to decrease inflammation. They are thought to be effective in treating mild-to-moderate episodes of Crohns disease[17] and useful as a maintenance treatment in preventing relapses of the disease. They work best in the colon and are not particularly effective if the disease is limited to the small intestine. These drugs have been widely used in the past but now are generally considered of limited benefit. Corticosteroids: they are a class of anti-inflammatory drug that are used primarily for treatment of moderate to severe flares of Crohn's disease. Also known as steroids, they are used more sparingly due to the availability of effective treatments with fewer side-effects.[18]. Corticosteroids help reduce the activity of the immune system and decrease inflammation. Health care providers prescribe corticosteroids for people with moderate to severe symptoms. Corticosteroids include Prednisone (the most commonly prescribed oral steroid), Budesonide, Hydrocortisone and Methylprednisone. They can help reduce inflammation anywhere in the body, but they have numerous side effects, including a puffy face, excessive facial hair, night sweats, insomnia and hyperactivity. More-serious side effects include high blood pressure, diabetes, osteoporosis, bone fractures, cataracts, glaucoma and increased chance of infection.

Also, corticosteroids don't work for everyone with Crohn's disease. Doctors generally use them only if you don't respond to other treatments. Budesonide (Entocort EC), a newer type of corticosteroid, works faster than do traditional steroids and appears to produce fewer side effects. However, it is only effective for Crohn's disease that's in certain parts of the bowel. It is also useful when used in combination with antibiotics for active Crohns disease.[19] Steroid enemas can also be used for disease of the lower colon and rectum, in order to treat symptoms. Hydrocortisone and budesonide liquid and foam enemas are being marketed for these reasons.Corticosteroids aren't for long-term use, but they can be used for short-term (three to four months) symptom improvement, to induce remission and should not be used as a maintenance medication. If a patient cannot come off steroids without suffering a relapse of his/her symptoms, the doctor may need to add some other medications to help manage the disease. Corticosteroids may also be used with an immune system suppressor the corticosteroids can induce remission, while the immune system suppressors can help maintain it.Immune system suppressors

This class of medications modulates or suppresses the bodys immune system response so it cannot cause ongoing inflammation. Immunomodulators generally are used in people for whom aminosalicylates and corticosteroids havent been effective or have been only partially effective. They may be useful in reducing or eliminating the need for corticosteroids. They also may be effective in maintaining remission in people who havent responded to other medications given for this purpose. Immunomodulators may take several months to begin working.

For some people, a combination of these drugs works better than one drug alone. Immunosuppressant drugs include:

Azathioprine (Imuran) and mercaptopurine (Purinethol): they are the most widely used immunosuppressants for maintenance therapy of Crohons disease. Taking them requires that the patient follow up closely with the doctor and have blood checked regularly to look for side effects, such as a lowered resistance to infection. Short term, they also can be associated with inflammation of the liver or pancreas and bone marrow suppression. Long term, although rarely, they are associated with certain infections and cancers including lymphoma and skin cancer. They may also cause nausea and vomiting. The doctor will use a blood test to determine whether you can take these medications.

Methotrexate (Rheumatrex): a folate anti-metabolite drug which is also used for chemotherapy, and it is useful in maintenance of remission for those no longer takinh corticosteroids.[20] This drug, which is used to treat cancer, psoriasis and rheumatoid arthritis, is sometimes used for people with Crohn's disease who don't respond well to other medications. Short-term side effects include nausea, fatigue and diarrhea, and rarely, it can cause potentially life-threatening pneumonia. Long-term use can lead to bone marrow suppression, scarring of the liver and sometimes to cancer. There is need to be followed closely for side effects.

Cyclosporine (Gengraf, Neoral, Sandimmune) and Tacrolimus (Astagraf XL, Hecoria): these potent drugs, often used to help heal Crohn's-related fistulas, are normally reserved for people who haven't responded well to other medications. Cyclosporine has the potential for serious side effects, such as kidney and liver damage, seizures, and fatal infections. These medications aren't for long-term use.

Ustekinumab (Stelara): this drug is used to treat psoriasis. Studies have shown it's useful in treating Crohn's disease as well and may be used when other medical treatments fail.

Antibiotics

Antibiotics may be used when infections, such as abscesses, occur in Crohns disease. They can also be helpful with fistulas around the anal canal and vagina. Antibiotics used to treat bacterial infection in the GI tract that may play a role in activating the intestinal immune system, leading to inflammation. They may be used in addition to other medications or when infection is a concern, such as with perianal Crohn's disease. However, there's no strong evidence that antibiotics are effective for Crohn's disease. Frequently prescribed antibiotics include:

Metronidazole (Flagyl): at one time, it was the most commonly used antibiotic for Crohn's disease. However, it can cause serious side effects, including numbness and tingling in your hands and feet and, occasionally, muscle pain or weakness. If these effects occur, the patient must stop the medication and call the doctor.

Ciprofloxacin (Cipro): this drug, which improves symptoms in some people with Crohn's disease, is now generally preferred to metronidazole. A rare side effect is tendon rupture, which is an increased risk if you're also taking corticosteroids.Metronidazole and ciprofloxacin are antibiotics which are used to treat Crohn's that have colonic or perianal involvement, although this use has not been approved by the Food and Drug Administration.[21] They are also used for treatment of complications, including abscesses and other infections accompanying Crohn's disease.[18]Biologic Therapies

These medications represent the latest class of therapy used for people with Crohn's disease who have not responded well to conventional therapy. These medications are antibodies grown in the laboratory that stop certain proteins in the body from causing inflammation. Infliximab (Remicade), Adalimumab (Humira) and Certolizumab pegol (Cimzia): These drugs, called TNF inhibitors or biologics, work by neutralizing an immune system protein known as tumor necrosis factor (TNF). They are used for adults and children with moderate to severe Crohn's disease to reduce signs and symptoms. They also may induce remission. In order to compare their benefits, researchers continue to study upon these drugs. TNF inhibitors may be used soon after diagnosis, particularly if the doctor suspects that the person is likely to have more severe Crohn's disease or a fistula. Sometimes they are used after other drugs have failed. They also may be combined with an immunosuppressant in some people, but this practice is somewhat controversial.

People with certain conditions can't take TNF inhibitors. Tuberculosis and other serious infections have been associated with the use of immune-suppressing drugs. The patient must talk to the doctor about the potential risks and have a skin test for tuberculosis, a chest X-ray and a test for hepatitis B before starting these medications. They are also associated with certain cancers, including lymphoma and skin cancers.

Natalizumab (Tysabri) and Vedolizumab (Entyvio): these drugs work by stopping certain immune cell molecules -integrins - from binding to other cells in the intestinal lining. Natalizumab is approved for people with moderate to severe Crohn's disease with evidence of inflammation who aren't responding well to any other medications.[22] A total of 3 large randomized controlled trials have demonstrated that natalizumab is effective in increasing rates of remission[23] and maintaining symptom-free status[24] in patients with Crohn's disease. Natalizumab has also been linked to PML (though only when used in combination with interferon beta-1a).[25] The label also recommends monitoring of liver enzymes due to concerns over possible damage or failure. Because the drug is associated with a rare but serious risk of progressive multifocal leukoencephalopathy (brain infection leading to death or severe disability), the patient must be enrolled in a special restricted distribution program to use it.

Vedolizumab recently was approved for Crohn's disease. It works like natalizumab but appears not to carry a risk of brain disease.Strucrure of Pharmaceutical drugs

Figure 1 - 5-Aminosalicylate (Mesalamine), Sulfasalazine, Balsalazide and Olsalazine

Figure 2 - Prednisone

Figure 3 Budesonide

Figure 4 Hydrocortisone

Figure 5 Methylprednisone

Figure 6 Azathioprine

Figure 7 Mercaptopurine

Figure 8 - Methotrexate

Figure 9 Cyclosporine

Figure 10 Tacrolimus

Figure 11 Ustekinumab

Figure 12 Metronidazole

Figure 13 Ciprofloxacin

The Role of HPLC in Drug AnalysisThe most characteristic feature of the development in the methodology of pharmaceutical and biomedical analysis during the past 25 years is that HPLC became undoubtedly the most important analytical method for identification and quantification of drugs, either in their active pharmaceutical ingredient or in their formulations during the process of their discovery, development and manufacturing.[26]-[32]

Figure 14 - Steps in a drug development and manucacturing in the pharmaceutical industry

The various stages of the pharmaceutical streamline of drug discovery, development and manufacturing, are summarized in Figure 14. Drug development starts with the discovery of a molecule with a therapeutic value.[33],[34] This can be done by high throughput screening during which separations by fast or ultra-fast HPLC are performed.[35] At the discovery stage there can be also characterizing synthetic or natural products.[36]-[39] Drug metabolism and pharmacokinetics (DMPK) is the step where the candidate compounds for drug are tested for their metabolism and pharmacokinetics. The studies involve use of LC-MS or LC-MS/MS.[40]-[43]The next stage is the development stage, where HPLC is used to characterize products of the chemical synthesis, by analyzing the active pharmaceutical ingredients (API), their impurities and/or degradation products generated by accelerated aging.[44] The development of formulation requires also studies of the dissolution properties of solid dosage forms as well as assays of the pharmaceutical formulations.[45] Method for the verification of system's cleanliness during the manufacturing process are developed and used at this stage.[46] All the HPLC methods that have been finalized at the developmental stage are validated and transferred to the manufacturing laboratories for a quality control analysis.[47]-[53]HPLC

Assay of Corticosteroids

A mixture of six corticosteroids namely: deoxycortisone, hydrocortisone acetate, cortisone, prednisone, hydrocortisone and prednisolone can be assayed by HPLC method. Following are presented the chromatographic parameters for the assay:Sample size: 10 L

Column: size-250 x 4.6 mm ID

Adsorbent: Lichrosorb DIOL: 10m

Mobile-phase: Gradient elution of A (n-Hexane) and B (Isopropanol)

Detector: UV-254 nm

The six well-elaborated and distinct peaks under the above HPLC parameters, are clearly shown in Figure 15.

Figure 15 - HPLC Chromatogram of CorticosteroidsBudesonide

The methods available for determination of Budesonide, as per literature, include Uv spectroscopy [54], Stability indicating HPLC [55], LC/MS method [56],[57] and TLC with UV detection [58]. One UV spectroscopy [59] and HPLC [60] methods were also reported for simultaneous determination of Budesonide in its combine dosage form. One LC/MS/MS method was reported as well for determination of Budesonide in human sputam sample.[61].Simultaneous HPLC analysis was also reported for determination of Budesonide along with triamcinolone acetonide in microdialysate and rat plasma. [62] Stability indicating LC [63] and visible spectroscopy.[64] RP-HPLC is found to be the most effective and widely used method where sensitivity as well as accuracy is concerned. Considering the wide importance of the novel steroids in the treatment of respiratory disorders, we developed simple, sensitive, accurate as well as precise RP-HPLC method for quantification of pure steroids and application of the developed method for determination of their metered dose commercial formulations were carried out. After development of the method, it was successfully validated as per validation parameters of ICH guidelines.[65]Budesonide, was soluble in methanol; therefore methanol was selected as common solvent for this steroid. Attempts were made to optimize a common mobile phase for the novel steroid which would give fast retention as well as detection at lower concentration. Various mobile phases were tried and retention time as well as peak area and peak symmetry was observed. Best results were obtained using Methanol: ACN in the ratio of 60: 40 v/v as mobile phase using C-18 column. The retention time (RT) of Budesonide was found to be 3.41610.1305. The selected mobile phase gives best result in terms of faster retention, least tailing of the peaks as well as accurate results for each kind of chromatographic measurements throughout the development of the entire method. The chromatogram of Budesonide was represented in Figure 16.

Figure 16 - HPLC Chromatogram of Budesonide1. NDDIC National Digestive Diseases Information Clearinghouse (2014). Crohns disease, July 10, 2013. Retrieved 12 June 20142. Crohn BB, Ginzburg L, Oppenheimer GD (2000). Regional ileitis: A pathologic and clinical entity. 1932, The Mount Sinai journal of medicine, New York 67 (3): 2638. PMID 108289113. Baumgart DC, Sandborn WJ; Sandborn (2012). Crohn's disease, The Lancet 380 (9853): 1590605. doi:10.1016/S0140-6736(12)60026-9. PMID 229142954. Dessein R, Chamaillard M, Danese S (2008). Innate Immunity in Crohns Disease. Journal of Clinical Gastroenterology 42: S1447. doi:10.1097/MCG.0b013e3181662c90. PMID 188067085. Stefanelli T, Malesci A, Repici A, Vetrano S, Danese S (2008). New Insights into Inflammatory Bowel Disease Pathophysiology: Paving the Way for Novel Therapeutic Targets, Current Drug Targets 9 (5): 41386. Cho JH, Brant SR (2011). Recent Insights into the Genetics of Inflammatory Bowel Disease, Gastroenterology 140 (6): 170412, doi:10.1053/ j.gastro.2011.02.046. PMID 215307367. Marks DJ, Rahman FZ, Sewell GW, Segal AW (2010). Crohn's disease: An immune deficiency state, Clinical reviews in allergy & immunology 38 (1): 2031. doi:10.1007/s12016-009-8133-2. PMID 194371448. Casanova JL, Abel L (2009). Revisiting Crohn's disease as a primary immunodeficiency of macrophages, The Journal of experimental medicine 206 (9): 183943. doi:10.1084/jem.20091683. PMID 196872259. Lalande JD, Behr MA (2010). Mycobacteria in Crohn's disease: How innate immune deficiency may result in chronic inflammation,. Expert review of clinical immunology 6 (4): 63341. doi:10.1586/eci.10.29. PMID 2059413610. Cosnes J (2004). Tobacco and IBD: Relevance in the understanding of disease mechanisms and clinical practice. Best Practice & Research Clinical Gastroenterology 18 (3): 4819611. Molodecky, NA; Soon, IS; Rabi, DM; Ghali, WA; Ferris, M; Chernoff, G; Benchimol, EI; Panaccione, R; Ghosh, S; Barkema, HW; Kaplan, GG (2012). Increasing incidence and prevalence of the inflammatory bowel diseases with time, based on systematic review. Gastroenterology 142 (1): 4654.e42; quiz e3012. Prideaux, L; Kamm, MA; De Cruz, PP; Chan, FK; Ng, SC (2012). Inflammatory bowel disease in Asia: a systematic review. Journal of Gastroenterology and Hepatology 27 (8): 126680. doi:10.1111/j.1440-1746.2012.07150.x. PMID 2249758413. Burisch, J; Munkholm, P (2013). Inflammatory bowel disease epidemiology. Current opinion in gastroenterology 29 (4): 35762. doi:10.1097/MOG.0b013e32836229fb. PMID 2369542914. Hovde, ; Moum, BA (2012). Epidemiology and clinical course of Crohn's disease: results from observational studies. World journal of gastroenterology: WJG 18 (15): 172331. doi:10.3748/wjg.v18.i15.1723. PMC 3332285. PMID 2255339615. Lozano, R; Naghavi, M (2012). Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet 380 (9859): 209512816. Hanauer, Stephen B.; Sandborn, William; Practice Parameters Committee of the American College of Gastroenterology (2001). Management of Crohn's disease in adults. The American Journal of Gastroenterology 96 (3): 63543. doi:10.1111/j.1572-0241.2001.03671.x. PMID 1128052817. Hanauer, S; Strmberg, U (2004). Oral pentasa in the treatment of active Crohn?s disease: A meta-analysis of double-blind, placebo-controlled trials,. Clinical Gastroenterology and Hepatology 2 (5): 37988. doi:10.1016/S1542-3565(04)00122-3. PMID 1511897518. Gopal, Latha; Nachimuthu, Senthil (2011). Katz, Julian, ed. Crohn Disease WebMD. Retrieved July 7, 201219. Steinhart, A; Feagan, BG; Wong, CJ; Vandervoort, M; Mikolainis, S; Croitoru, K; Seidman, E; Leddin, DJ et al. (2002). Combined budesonide and antibiotic therapy for active Crohn's disease: A randomized controlled trial. Gastroenterology 123 (1): 3340. doi:10.1053/gast.2002.34225. PMID 1210583120. Ursing, B; Alm, T; Brny, F; Bergelin, I; Ganrot-Norlin, K; Hoevels, J; Huitfeldt, B; Jrnerot, G et al. (1982). A comparative study of metronidazole and sulfasalazine for active Crohn's disease: The cooperative Crohn's disease study in Sweden. II. Result. Gastroenterology 83 (3): 5506221. Feagan, Brian G.; Fedorak, Richard N.; Irvine, E. Jan; Wild, Gary; Sutherland, Lloyd; Steinhart, A. Hillary; Greenberg, Gordon R.; Koval, John et al. (2000). A Comparison of Methotrexate with Placebo for the Maintenance of Remission in Crohn's Disease. New England Journal of Medicine 342 (22): 16273222. Michetti, Pierre; Mottet, Christian; Juillerat, Pascal; Pittet, Valrie; Felley, Christian; Vader, John-Paul; Gonvers, Jean-Jacques; Froehlich, Florian (2007). Severe and Steroid-Resistant Crohn's Disease. Digestion 76 (2): 99108. doi:10.1159/000111023. PMID 1823940023. Ghosh, Subrata; Goldin, Eran; Gordon, Fiona H.; Malchow, Helmut A.; Rask-Madsen, Jrgen; Rutgeerts, Paul; Vyhnlek, Petr; Zdorov, Zdena et al. (2003). Natalizumab for Active Crohn's Disease. New England Journal of Medicine 348 (1): 2432. doi:10.1056/NEJMoa020732. PMID 1251003924. Feagan, Brian G.; Sandborn, William J.; Hass, Steven; Niecko, Timothy; White, Jeffrey (2007). Health-Related Quality of Life During Natalizumab Maintenance Therapy for Crohn's Disease. The American Journal of Gastroenterology 102 (12): 273746. doi:10.1111/j.1572-0241.2007.01508.x. PMID 1804210625. Kleinschmidt-Demasters, B.K.; Tyler, Kenneth L. (2005). Progressive Multifocal Leukoencephalopathy Complicating Treatment with Natalizumab and Interferon Beta-1a for Multiple Sclerosis. New England Journal of Medicine 353 (4): 36974. doi:10.1056/NEJMoa051782. PMID 1594707926. Schirmer, R. (1991). Modern Methods of Pharmaceutical Analysis, CRC Press27. Tang, L., Fitch, W., Alexander, M., Dolan, J. (2000). Anal. Chem 72, 5211-521828. Lee, D., Webb, M. (2003). Pharmaceutical Analysis, Blackwell Science Ltd29. Lunn, G., Wiley, J. (2005). HPLC methods for recently approved pharmaceuticals, Wiley-Interscience30. Gorog, S. (2006). Trends in Analytical Chemistry 25, 755-75731. Gorog, S. (2007). Trends in Analytical Chemistry 26, 12-1732. Kazakevich, Y. V., LoBrutto, R. (2007). HPLC for Pharmaceutical Scientists, Wiley-Interscience

33. Bleicher, K., Bohm, H., Muller, K., Alanine, A. (2003). Nature Reviews Drug Discovery 2, 369-37834. Daniel, B. (2007). HPLC for Pharmaceutical Scientists35. Castro-Perez, J., Plumb, R., Granger, J. H., Beattie, I., Joncour, K., Wright, A. (2005). Rapid Commun. Mass Spectrom 19, 84384836. Butler, M. S. (2004). Journal of Natural Products 67, 2141-215337. Smyth, W. F., McClean, S., Hack, C. J., Ramachandran, V. N., Doherty, B., Joyce, C., ODonnell, F., Smyth, T. J., OKane, E., Brooks, P. (2006). Trends in Analytical Chemistry 25, 572-58238. Butler, M. S. (2005). Natural Product Reports 22, 162-19539. Lam, K. S. (2007). Trends in Microbiology 15, 279-28940. Chu, I., Anomeir, A. (2006). Current Drug Metabolism 7, 467-47741. Kassel, D. B. (2005). Using Mass Spectrometry for Drug Metabolism Studies42. Lappin, G., Garner, R. C. (2005). Expert Opin. Drug Metab. Toxicol. 1, 23-3143. Kassel, D. B. (2004). Current Opinion in Chemical Biology 8, 339-34544. Bakshi, M., Singh, S. (2002). Journal of Pharmaceutical and Biomedical Analysis 28, 1011-104045. Rosanske, T. W., Brown, C. K., Christopher, M.R.a.T.W.R. (1996) in Progress in Pharmaceutical and Biomedical Analysis, Vol. Volume 3, pp. 169, Elsevier46. Gomis, D. B., Nunez, N. S., Enguita, P. B., Abrodo, P. A., Alvarez, M. D. G. (2006). Journal of Liquid Chromatography & Related Technologies 29, 931-94847. Rozet, E., Dew, W., Morello, R., Chiap, P., Lecomte, F., Ziemons, E., Boos, K. S., Boulanger, B., Crommen, J., Hubert, P. (2008). Journal of Chromatography A 1189, 32-4148. Koh, H. L., Yau, W. P., Ong, P. S., Hegde, A. (2003). Drug Discovery Today 8, 889-89749. Wrezel, P. W., Chion, I., Hussain, M. S. (2004). LC GC NORTH AMERICA 22, 1006-100950. Brandt, A., AG, S., Berlin, G., Kueppers, S. (2002). LC GC Europe March, 251. Varma, S. (2001). The Quality Assurance Journal 5, 85-9252. Jimidar, M. I., De Smet, M. (2007). HPLC Method Development for Pharmaceuticals53. Gibson, M. (2005). Technology Trandfer Introduction and Objectives, PDA54. Sanap, D. D., Sisodia, A. M., Patil, S. H., and Janjale, M. V. (2011). Novel And Validated Spectrophotometric Determination Of Budesonide From Bulk And Tablets Using Mixed Hydrotropic Solubilization Technique. International journal of Pharmaceutical science and research, 2, 9: 2419-242355. Hou, S., Hindle, M., Byron, P. R. (2001). A stability-indicating HPLC assay method for Budesonide. Journal of Pharmaceutical and Biomedical Analysis, 24: 371-38056. Deventer, K., Mikulikov, P., Hoecke, H. V., Eenoo, P. V., and Delbeke, F. T. (2006). Detection of budesonide in human urine after inhalation by liquid chromatography-mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis, 42: 474-47957. Lindberg, C., Blomqvist, A., Paulson, J. (2005). Determination of (22R,S) budesonide in human plasma by automated liquid chromatography/thermospray mass spectrometry. Biological Mass Spectrometry, 21, 11: 525-53358. Krzek, J., Hubicka, U., Dabrowska, M., Leciejewicz E. (2002). Determination of budesonide R(+) and S() isomers in pharmaceuticals by thin-layer chromatography with UV densitometric detection. Chromatographia, 56, 11: 759-76259. Prasad, A. V. S. S. (2006). Simultaneous spectrophotometric estimation of budesonide and formoterol fumarate in their combined dosage forms. Indian journal of chemical technology, 13: 81-8360. Assi, K. H., Tarsin, W., Chrystyn, H. (2006). High performance liquid chromatography assay method for simultaneous quantitation of formoterol and budesonide in Symbicort Turbuhaler. Journal of Pharmaceutical and Biomedical Analysis, 41: 325-32861. Buscher, B. A., Jagfeldt, H., Sandman, H., Brust-van S. R., Schaik, F., Brll, L. P. (2012). The determination of budesonide and fluticasone in human sputum samples collected from COPD patients using LC-MS/MS. Journal of Chromatography B Analytical Technology and Biomedical Life Science, 880, 1: 6-1162. Vieira, M. L. T., Singh, R. P., Derendorf, H. (2010). Simultaneous HPLC analysis of triamcinolone acetonide and budesonide in microdialysate and rat plasma: Application to a pharmacokinetic study. Journal of Chromatography B, 878: 2967-297363. Maximiliano, S., Daniele, R. N., Luclia, M., Diogo, P. L., and Sergio, L. D. (2008). Validation of a Stability Indicating Reversed Phase LC Method for the Determination of Fluticasone Propionate in Pharmaceutical Formulations. Journal of Liquid Chromatography and Related Technologies, 31: 2113-212764. Ayad, M. M., Henawee, M. M., Abdellatef, H. E., Sayed, H. (2005). Spectrophotometric determination of levonorgestrel, norethisterone acetate, mometasone furoate and fluticasone propionate using 4 amino-antipyrine, 43, 3: 24-2865. International Conference on Harmonization (ICH) of Technical Requirements for the Registration of Pharmaceutical for Human Use, Validation of Analytical Procedures: Text and Methodology Q2 (R1). 2005: 1-13PAGE 1