Module 6

Module 6 Quality Assurance/Quality Control Microbiology and Public Health, Qualitative Pharmaceutical Chemistry, Drug Testing with Instrumentation

HISTORY  Robert Hooke  Anton van Leeuwenhoek

- cell theory - Father of Microbiology; “animalcules”

Francesco Redi

Opposed Theory of Spontaneous Generation

John Needham

Theory of Spontaneous Generation

Lazzaro Spallanzani

To Needham: “Microbes entered after boiling because your flask is open!” From Needham: “Vital force was lost because you covered your flask, you bastard.”

Anton Laurent Lavoisier

Oxygen is important to life To Spallanzani: “There was not enough Oxygen in the flask to support life!”

Rudolf Virchow

Proposed Theory of Biogenesis: ‘Omni Cellula Celluli’

Louis Pasteur

‘Microorganisms are present in air, but air itself does not create microbes’ (Pasteur Flask) Coined “vaccine” (developed immunity for anthrax, rabies, cholera) Fermentation, Pasteurization

Joseph Lister Robert Koch

RichardPetri Ignaz Semmelweiz MODERN CHEMOTHERAPY o Paul Ehrlich o Emil von Behring o Domaqk o AlexanderFleming o Florey andChain o Selman Waksman o Edward Jenner

Father of Antiseptic Surgery (Phenol/Carbolic acid) Father of Microbiological Techniques, Koch Postulate, Germ Theory  M. tuberculosis (Koch’s bacilli)  B. anthracis (biggest)  V. cholerae (comma shaped halophile) Surgical handwashing to prevent Puerperal Childbirth Fever

- Father of Chemotherapy (Salvarsan) - Diphtheria toxin - Prontosil - Penicillum notatum - Penicillin - Streptomycin - smallpox vaccine

5 Kingdom Classification System (Whittaker): Monera, Protista, Fungi, Plantae, Animalia Kathreen Mae Doria Cascabel

Prokaryotic Nucleus Nuclear membrane Organelles DNA Cell wall Division Sexual Recombination Ribosomes

    

No No No No histones Peptidoglycan Binary fission None, DNA transfer only 70s

Eukaryotic Yes Yes Yes Linear, associated with histones None in animals and protozoa only Mitosis Meiosis 80s, 70s in organelles

CHO (starch, glycogen) CHON ( enzymes) Lipids Inorganic ions 80% water

Kapsule Cell W Oll

Pilus/Fimbriae

Polysaccharide or polypeptide Welch method/Negative staining - India ink or Nigrosin Peptidoglycan (NAG, NAM) Endotoxin(G-) - Lipoprotein (lipid A) - More heat stable than exo - MOA: ReleaseTNF Hair-like structures Pilin (protein)

FlageHum

Flagellin (protein)

Spore

Dipicolinic acid (heat resistance)

Evasion of phagocytosis Adherence/Attachment Resistance to dessication Shape Resistance to lysis

Streptococcus mutans Streptococcus pneumoniae Cryptococcus neoformans Listeria monocytogenes (only G+ that has endotoxin)

Adherence (fimbriae) Conjugation (pilus) Virulence Motility Chemotaxis Run and tumble movement Survival Heat and chemical resistance

Neiserria gonorrhea (virulence) Listeria monocytogenes (tumbling motility)

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BACTERIAL PHYSIOLOGY Generation Time – time for bacteria to double in size and split into two Growth Curve 1. Lag Phase(0) 2. Log Phase/Exponential Growth Phase (1) 3. Stationary Phase (2) 4. Death Phase/Logarithmic Decline Phase (3)

PHYSICAL REQUIREMENTS 1. Temperature Psychrophiles Psychrotrophs Mesophiles Thermophiles Hyperthermophiles

- cold loving (0°C) - grow in refrigerator (20-30°C) - moderate (25-40°C) - heat loving (50-60°C) - extreme thermophiles (80°C)

*Minimum growth temperature, Maximum growth temperature, Optimum growth temperature 2. pH (Optimum: 6.5-7.5) Acidophiles, Alkaliphiles, Neutrophile 3. Osmotic Pressure/Salt Concentration Extreme halophiles - require high concentration for growth Obligate halophiles - grows at extremely high concentration (nearly 30%) Facultative halophiles - require high salt but can grow at concentrations up to 2%

CHEMICAL REQUIREMENTS 1. Carbon Chemoheterotrophs - get C from their source of energy (CHON, CHO, lipids) Chemoautotrophs & Photoautotrophs - get C from carbon dioxide 2. N, S, P, Trace elements 3. Oxygen

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CULTURE MEDIA  Nutrient material prepared for microbial growth Agar - complex polysaccharide derived from marine algae Colony - visible growth of microbes on the surface of a medium Chemically Defined Simple Complex Enriched Anaerobic/Reducing Selective

Differential Combined Selective and Differential

Exact chemical composition is known For non-fastidious organisms Made up of nutrients from diff sources To increase very small numbers to detectable levels; for fastidious For obligate anaerobes Suppress growth of unwanted & encourage the growth of desired ones

Distinguish colonies on same plate

Neisseria gonorrhea

Milk agar Blood agar Thioglycolateagar Bismuth sulphite agar Saboraud’s dextrose agar Brilliant green agar (Salmonella) Middlebrook (Mycobacterium) Lowenstein-Jennsen (Mycobacterium) Colistin, Nalidixic acid (g +) Blood agar (Streptococci α, β, γ) Mannitol salt agar (S.aureus) MacConkey agar

CULTURE MEDIA 1. 2. 3. 4. 5. 6. 7. 8. 9.

SDA/PDA LJ Loeffler’s TCBS Skirrow’s Chocolate/Blood L-cysteine Thayer-Martin VCN Fletcher’s

- fungi - M. tuberculosis - diptheriae - cholera - H. pylori, C. jejuni - H. influenza - Legionella pneumophila - Neisseria - Leptospira interrogans

STAINING METHODS 1. 2. 3. 4. 5.

Gram staining Schauffer-Fulton Acid Fast/Ziehl Neelsen Feulgen Welch/Negative

- G+ (B/V), G- (R/P) - Spore formers (M. green) - Mycobacterium, Nocardia - Nuclei/DNA - Capsule (Nigrosin/India Ink)

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GRAM POSITIVES (DRUG OF CHOICE: PENICILLIN G) COCCI DISEASE Streptococcus Pneumoniae Adult pneumonia (-) catalase Otitis media (DOC: Cefaclor) Adult meningitis α – pneumoniae, Pyogenes (GABHS) Pharyngitis/sore throat viridans Skin infections Scarlet fever β – pyogens (A),

MISCELLANEOUS Test: Quellung Reaction & Optochin Antibody mediated (DOC: Pen G + pred) Rheumatic Heart Fever Glomerulonephritis

agalactiae (B)

Staphylococcus (+) catalase

Viridans Agalactiae (GBBHS) Aureus (+)coagulase

Epidermidis Saprophiticus

BACILLI

Spore forming

Nonspore forming

Subacute bacterial endocarditis Neonatal meningitis and pneumonia From exotoxin release: 1. Gastroenteritis 2. TSS (+) T cells  cytokines 3. SSS By direct organ invasion: 1. Pneumonia, Meningitis, Cerebritis 2. Osteomyelitis 3. Acute endocarditis 4. Septic arthritis 5. Skin infections Catheter 2nd cause of UTI in sexually activewomen

Halophilic (15% NaCl)

Coagulase (-) Coagulase (-)

Bacillus anthracis

DISEASE Anthrax, Woolsorter’s disease

Capsule: poly-D-glutamate

TREATMENT Pen G Ciprofloxacin

Bacillus cereus

Diarrhea

From fried rice

Pen G

Clostridium botulinum

Flaccid paralysis (Fluffy Baby Syndrome)

Blocks Ach release

Metronidazole

Clostridium tetani

Spastic paralysis

Blocks GABA & glycine

Metronidazole

Clostridium perfringes

Gas gangrene (Clostridial myonecrosis)

α-toxin/lecithinase: splits lecithin into phosphocholine & diglyceride

Clostridium difficile

Pseudomembranous colitis caused by Clindamycin

Metronidazole Clindamycin Pen G Metronidazole Vancomycin

Inh. elongation factor 2 Screening: Schick test, Diagnosis: Loeffler’s slant Tumbling motility Only g+ with endotoxin

DPT Vaccine Erythromycin Pen G Cotrimoxazole Ampicillin

Corynebacterium diphtheriae

Diphtheria

Listeria monocytogenes

Common cause of meningitis in neonates

MISCELLANEOUS

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GRAM NEGATIVES

ENTEROBACTERIACEAE Salmonella spp.  Motile, Produce H2S S. typhi   

Non-lactosefermenter Form acid in glucose & mannose

S. choleraesius S. enteritidis Proteus mirabilis (Swarming motility) Mostly in turtle & snake eggs

Escherichia coli   

Normally found in GI UTI, meningitis, sepsis Common cause of HAP

Klebsiella pneumoniae  

Present in respiratory tract and feces Nonmotile

Shigella dysenteriae   

DISEASE Typhoid fever Rotten egg diarrhea Rose Spots on the belly Sepsis Diarrhea/Gastroenteritis Common cause of UTI and nosocomial infections

TREATMENT Chloramphenicol Ceftriaxone(NEW)

ETEC EPEC EHEC EIEC EAEC

Cotrimoxazole Quinolones

Traveller’s/Montezuma’s Children’s Bloody (no pus & fever) Bloody (with pus & fever) Watery diarrhea

2nd most common causeof G(-) sepsis Dysentery

Cotrimoxazole Quinolones

Non-motile, Non-lactose fermenter Non H2S producer Shiga toxin: inhibits 60s ribosomes

VIBRIONACEAE 



DISEASE

TREATMENT

Oxidase (+), polar flagella Require saline or alkaline media (Thiosulfate-citrate bile salt-sucrose agar/TCBS)

Exotoxin: Cholera toxin (increases cAMP levels) Contaminated raw fish (sushi); halophilic Zoonotic; from uncooked poultry, unpasteurized milk Spiral, highly motile, microaerophile, Urease (+)

Vibrio cholera (comma-shaped) V. parahemolyticus Campylobacter jejuni

Helicobacter pylori

NEISSERIAE   

“rice watery diarrhea”

Tetracycline, ORS

Bloody diarrhea

Quinolones

Ulcer

TeAM B (Tetra, Amox, Metro, Bismuth) ClarO (Clarithro + Ome) DISEASE

DOC

Nonmotile diplococcus (kidney bean) Oxidase (+), ferment CHO (acid) Medium: Thayer Martin VCN

Neisseria meningitidis

Glucose and maltose fermenter Virulence: Capsule/Glycocalyx

Meningitis Meningococcemia

Ceftriaxone

Neisseria gonorrheae

Ferments glucose only Virulence: Pili/adhesins, IgA Protease, Endotoxin

Gonorrhoea, STD, PID (Pelvic Inflammatory Disease)

Ceftriaxone

Gonococcal Ophthalmia Neonatorum

AgNO3 (old), Erythromycin (new)

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Aerobe

P.aeruginosa Oxidase (+)

Anaerobe

Bacterioides fragilis

Pneumonia Opportunistic (burn patients) 1° cause of nosocomial infections GIT abscesses

Exotoxin A (diphtherialike); inhibits EF2

Anti pseudomonal penicillins and cephalosporins

Normal flora of the colon (most predominant)

Metronidazole, Clindamycin, Chloramphenicol

ATIPYCAL

DISEASE

VECTOR

Rocky Mountain Spotted Fever Damages endothelial cells (vessel) Rickettsial pox

Wood/Dog tick

Human Louse

typhi

Endemic typhus Brill-Zinsser disease Endemic typhus

tsutsugamushi

Scrub typhus

Mite larvae

quintana

Trench fever

Body louse

henselae

Cat scratch disease

trachomatis

Blindness (trachoma)

psittaci

Parrot fever

DOC: Tetracycline, Chloramphenicol Rickettsia rickettsii  Pleomorphic, Peptidoglycan akari has muraminic acid & diaminopimelic acid prowazekii 

Growth enhanced by sulphonamides

Bartonella

Chlamydia

RESPIRATORY TRACT

Mites

Rat flea

DISEASE

TREATMENT

Haemophilus influenzae

Encapsulated Lab: Chocolate agar

HIB: Meningitis in children

2 and 3rd gen ceph HIB vaccine

Haemophilus ducreyi

Sexualtransmission

Chancroid

Erythromycin, Ceftriaxone

Haemophilus vaginalis

Sexualtransmission

Bacterial vaginitis Fishy vaginal discharge

Metronidazole

Whooping cough

DPT vaccine

Bordetella pertussis

nd

Legionella pneumophila

Aircons, showerheads

Legionnaire’s disease/Pontiac fever

Erythromycin, Rifampicin

Coxiella burnetti

Aerosols Contaminated milk

Q fever

Doxycycline, Chloroquine Kathreen Mae Doria Cascabel

ZOONOTIC Yersinia pestis

RESERVOIR

MOT

Rodents, squirrels

Flea bite, animal contact

Yersinia enterolitica

DISEASE

Unpasteurized milk

Francisella tularensis

Rabbits, squirrels

Pasteurella multocida

Tick, deerfly

Bubonic plague

Streptomycin (DOC)

Enterocolitis Arthritis Rash, Diarrhea Rabbit fever

ORS Fluoroquinolones Cotrimoxazole Streptomycin (DOC)

Dog/cat bites

Brucella melitensis B. abortus B. suis B. canis

TREATMENT

Penicillin

Goat Cattle Pig Dog

SPIROCHETES

MISC

CLINICAL

TREATMENT Pen G

Treponema pertenue

Syphilis (1°: painless chancre) Diagnosis: Dark field microscopy Yaws: severe facial disfigurement

Treponema carateum

Pinta: flat red or blue lesions

Pen G Doxycycline & Amoxicillin

Treponema pallidum

Microaerophilic

Borrelia burgdorferi

Vector: Ixodes ticks

Lyme disease

Borrelia recurrentis

Vector: louse

Relapsing fever

Leptospira interrogans

Culture: Fletcher’s media

Leptospirosis Weil’s disease

Pen G

Prophylaxis: Doxycycline DOC: Pen G

ACID-FAST BACTERIA  Rod shaped, aerobic, do not form spores, resist decolorization by acid or alcohol  Ziehl Neelsen Stain/Acid Fast Stain (+)red, (-)blue o Carbolfuschin, Heat, Acid alcohol, Methylene Blue

M. tuberculosis

MISC Cell wall: mycolic acid Media: LJ (serpentine colonies)

M. leprae Mycoplasma pneumonia Atypical mycobacteria

Smallest known free-living self replicating prokaryote M. avium-intracellulare M. kansasii M. marinum M. scrofulaceum M. ulcerans M. fortultum

CLINICAL Pulmonary tuberculosis

RIPES

TREATMENT

Leprosy/Hansen’s Disease (Lepromatous or Tuberculoid) Walking pneumonia

Dapsone, Rifampicin, Clofazimine TETRACYCLINE

- Infects AIDS patients - Pulmonary TB - Skin granuloma - Cervical lymphadenitis (Scrofula) - Chronic skin ulcers with necrotic centers - Skin abscesses, corneal ulcer, pulmonary infection Kathreen Mae Doria Cascabel

ANTIBACTERIAL AGENTS CELL WALL BETA-LACTAM ANTIBIOTICS 1. Penicillins (Penicillum notatum)- Interfere with last step of cell wall synthesis (transpeptidation or cross-linking) o Beta lactam attached to thiazolidine ring, Nucleus: 6-aminopenicillanic acid I.

Natural Penicillins Penicillin G (Benzylpenicillin) – PO, IV, IM(Procaine and Benzocaine) Use: RHD and Syphilis Penicillin V (Phenoxymethylpenicillin) – PO

II.

Penicillinase-Resistant/Antistaphylococcal Penicillins/Narrow Spectrum Methicillin (2,6-dimethoxyphenylpenicillin) - Prototype; off the market (interstitial nephritis) Nafcillin (2-ethocy-1-phenylpenicillin) Isoxazolyl Penicillins (Oxacillin, Cloxacillin,Dicloxacillin – best absorbed)

III.

Aminopenicillins/Broad Spectrum Ampicillin (Parenteral, poor GI absorption) - Prodrugs: Hetacillin, Bacampicillin, Cyclacillin Amoxicillin (PO)

IV.

Extended Spectrum/Anti-Pseudomonal Penicillins Carboxypenicillins (Carbenicillin, Ticarcillin) Ureidopenicillins (Piperacillin – most potent, Azlocillin, Mezlocillin)

2. Beta-lactamase Inhibitors (Clavulanic acid, sulbactam, tazobactam) o No antibacterial activity. They inactivate beta-lactamases 3. Cephalosporins o Beta-lactam ring attached to dihydrothiazine ring; Nucleus: 7-aminocephalosporanic acid Generation Gram+ GramFirst fa, pha, Cephradine +++ + Second fo, fu, fp, Cefaclor, Cefamandole, Loracarbef +++ ++ Third t/d, pera, tax, Moxolactam + +++ Fourth Cefepime, Cefpirome ++ ++++ Fifth Ceftaroline ++++ ++++ 2. Carbapenems Thienamycin, Meropenem, Imipenem (cleaved by dihydropeptidase; cilastatin) 5. Monobactam (Aztreonam) o The beta-lactam ring is not fused to another ring, no cross sensitivity with penicillin BACITRACIN (Bacillus subtilis)  MOA: inhibits mucopeptide cell wall synthesis of G (+) bacteria VANCOMYCIN (S. orientalis)  Glycopeptide  DOC for Pseudomembranous colitis caused by Clindamycin  Redman's syndrome (diphenhydramine before administering; slow IV infusion/drip) Kathreen Mae Doria Cascabel

PROTEIN SYNTHESIS Binds to 30s Ribosomes AMINOGLYCOSIDES (Streptomycin, Amikacin, Gentamicin, Netilmicin, Neomycin, Tobramycin) o “mycin” – derived from Streptomyces o “micin – derived from Microspora o AE: Ototoxicity, Nephrotoxicity, Neuromuscular blockade TETRACYCLINES (Doxycycline, Minocycline, Tetracycline, Demeclocycline) o Broadest spectrum antibiotic (gram (+), gram (-), spirochetes,mycoplasma, rickettsia, chlamydia) o Contain four fused rings with a system of conjugated double bonds o AE: Gastric discomfort, deposition in the bones and primary dentition causing discoloration and hypoplasia of the teeth and a temporary stunting of growth, hepatotoxicity, phototoxicity (demeclocycline), vestibular problems (minocycline) o Fanconi-like syndrome – ingestion of expired tertacycline o Chlortetracycline/Aureomycin (S. Aureofaciens)

Binds to 50s Ribosomes MACROLIDES o Common chemical characteristics 1. A large lactone ring 2. A ketone group 3. A glycosidically linked amino sugar o AE: epigastric distress, cholestatic jaundice (estolate form of erythromycin) Erythromycin (Ilotycin) - S. erythreus  Preferred drug for Legionnaire’s disease, Mycoplasma pneumoniae and Campylobacter, Chlamydial infections, Diphtheria, Pertussis  DOC for Pen G allergy Clarithromycin - Used with Omeprazole or Lansoprazole (H.pylori eradication) Azithromycin, OD - For nongonococcal urethritis (Chlamydia, LRTI, PID, pharyngitis, Legionnaire’s) LINCOSAMIDES (Clindamycin) - Streptomyces lincolnensis o 7-chloro-7-deoxylincomycin o Resemble sulfonamides in antibacterial spectrum and biochemical MOA o For abdominal and female genitourinary tract infections caused by B. fragilis o AE: Pseudomembranous colitis, rash, (DOC: Vancomycin) CHLORAMPHENICOL (S. venezuelae) o For typhoid fever (Ceftriaxone-new DOC), meningococcal infections and H.influenza infections in cephalosporin-allergic patients, anaerobic infections o Precautions and monitoring: i. Bone marrow suppression (dose-related) ii. Aplastic anemia (non-dose related) iii. Gray Baby Syndrome (neonates) Kathreen Mae Doria Cascabel

ALTERATION OF CELL MEMBRANE PERMEABILITY Polymyxin  Polymixin B and Colostin (Polymixin E)  Basic polypeptides  Cationic, surface-active compounds that disrupt the permeability of both outer and cytoplasmic membranes of gram(-) bacteria

INHIBITION OF NUCLEIC ACID SYNTHESIS Quinolones, Fluoroquinolones o Patterned after nalidixic acid (introduced for the treatment of UTI and URTI) o 1,4-dihydro-4-oxo-3-pyridinecarboxylic acid moiety (essential for antibacterial activity) o Fluoroquinolones have enhanced antibacterial activity (Ciprofloxacin is the most potent) o Chelates with metals o MOA: Inhibits DNA gyrase (topoisomerase II) and supercoiling o AE: Diarrhea, nausea, headache, dizziness, nephrotoxicity, phototoxicity

COMPETITIVE INHIBITION OF ENZYME ACTIVITY Antimetabolites

SULFONAMIDES o Gerard Domagk studied a bright dye, Prontosil (metabolized in vivo to sulfanilamide – active) o MOA: Compete with PABA for dihydropteroate synthetase  prevents synthesis of folic acid o AE: Crystalluria, Steven-Johnson Syndrome, Kernicterus, Anemia o Sulfonamides are usually used with dihydrofolate reductase inhibitors (e.g Trimethoprim) 1. DOC forUTI - Sulfamethoxazole and trimethoprim/Cotrimoxazole (Bactrim®) 2. DOC for Pneumocystis carinii - Bactrim; alternative drug: Pentamidine (aromatic diamide) 3. Burn therapy - Silver sulfadiazine and Mafenide (Flammazine®) 4. Conjunctivitis - Sodium sulfacetamide 5. Chloroquine-resistant malaria - Quinine + pyrimethamine + sulfadoxime (Fansidar®)

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GENERAL EUKARYOTIC STRUCTURES        

Molds Multicellular Can develop tiny spores that ride in the air and spread by air currents Reproduction: Sporulation Neutral pH, room temp Hyphae: ribbon-like structures Septae: Mycelium: intertwined mass Spores

    

Yeasts Unicellular Simplest form of fungi Reproduction: Budding Pseudohyphae Capsule: Negative stain (India ink)

Asexual: 1. 2. 3. 4. 5.

Sporangiospores Conidiospores Blastospores Arthrospores Chlamydospores

– sac – at tips/sides of hyphae – budding directly off a vegetative cell – septate hyphae secments at cross walls – thick wall

Sexual: 1. Zygospores 2. Basidiospores 3. Ascospores FungalDetection 1. Direct Microscopy 2. Antigen Detection 3. Culture

– diploid cell zygotes – club shaped cells – ascus

- KOH, calcofluor white, methenamine silver - Latex agglutination: Cryptococcus neoformans - Saboraud’s dextrose agar/PDA Yeast: sugar fermentation and assimilation biochemistry Mold: Macroscopic: colony morphology, pigmentation Microscopic: hyphae, conidia, sexual structures

Epidermophyton Microsporum Trichophyton

- skin & nails - hair & skin - hair, skin, nails

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SUPERFICIAL Black piedra

Piedra hortai

White piedra

Trichosporon beigelli

Pityriasis versicolor

Malasseiza furfur

Nodular infection of the shaft Black nodules like pebbles Larger, softer yellowish nodules on the hairs Axilla, pubic, beard, scalp Invasion of the cornified skin Hyper or hypopigmented maculae Lab diagnosis: skin scrapings (spag & meatballs)

Topical Imidazoles:  Clotrimazole  Tioconazole  Miconazole

CUTANEOUS Dermatophytoses

Tinea (Ringworms)

Candidiasis

Candida albicans

T. pedis (foot), T. capitis (head), T. corporis (body), T. cruris (genitalia), T. ungium (nails) Normal flora (can become opportunistic) Predisposing factors:  Vaginal candidiasis  Generalized candidiasis  Paronychia  Chronic mucocutaneous candidiasis

Griseofulvin

Nystatin

SUBCUTANEOUS Mycetoma Sporotrichosis Chromoblastomycosis

Eumycotic mycetoma Sporothrix schenckii Phialophora Cladosporium

Progressive painful swelling Necrotic, ulcerative nodules Found in rotting woods: Cauliflower-like warts

OralKI Systemic Triazoles Terbinafine

SYSTEMIC Blastomycosis

B. dermatitidis

North American Blastomycosis

Amphotericin B

Paracoccidiomycosis

P.brasilensis

South American Blastomycosis

Amphotericin B

Histoplasmosis “great mimic” Coccidiomycosis

H. capsulatum C. immitis

African Histoplasmosis Affects RES of macrophages in lungs Pulmonary infection (dark skinned type B male)

Amphotericin B Itraconazole Amphotericin B

Cryptococcosis

C. neoformans

Skin, CNS lesions

Fluconazole

Systemic Candidiasis

C. albicans

Disturbance in the normal flora

Amphotericin B

Aspergillosis

A. flavus A. fumigatus

Causes allergy, source of aflatoxin Farmer’s lung

Amphotericin B

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ANTIFUNGAL AGENTS A. AZOLES  MOA: Inhibits fungal CP450  inhibits ergosterol synthesis IMIDAZOLES  Miconazole  Toconazole  Clotrimazole  Ketoconazole

Ketoconazole, Itraconazole, Fluconazole -

Clotrimazole, Miconazole, Econazole -

TRIAZOLES  Itraconazole  Fluconazole

For SC and systemic mycoses

For superficial mycoses

- DOC for cryptococcal meningitis (Cryptococcus neoformans)

B. POLYENES MOA: Pore formation; binds to ergosterol present in the cell membrane disrupting membrane function, allowing electrolytes to leak out from the cell, resulting in cell death A. Amphotericin B

(Streptomyces nodosus)

B. Nystatin C. Natamycin

(Streptomyces noursei) (Streptomyces natalensis)

C. ALLYLAMINE 1. Terbinafine

- DOC for systemic mycoses

– inhibits squalene epoxidase

D. NUCLEOSIDES MOA: Inhibits DNA and RNA synthesis Flucytosine o Pyrimidine analog o Used only in combination with Amphotericin B for the treatment of systemic mycoses and meningitis caused by Cruptococcus neoformans and Candida E. GRISEOFULVIN (Penicillum griseofulvum)  MOA: Interacts with the microtubule within the fungus and inhibit mitosis (metaphase)  Absorption is increased with fatty acids  DOC for Dermatophytoses

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HELMINTHS

NEMATODES (ROUNDWORMS) INTESTINAL Ascaris lumbricoides

Intestinal roundworm

Ingest eggs

Necator americanus Ancylostoma duodenale Stronyloides stercoralis

New world hookworm Old world hookworm Threadworm

Skin penetration

Trichinella spiralis Trichuris trichura Enterobius vermicularis Capillaria philippinensis

Muscle roundworm Whipworm Pinworm Bagsit

Raw pork Ingest eggs Ingest eggs

Skin penetration

Eggs/adult worm in stool Fecal (eggs) Fecal (larvae) Enterotest

Scotch tape

Mebendazole, P,A Thiabendazole

Mebendazole, P,A

BLOOD AND TISSUE Onchocerca volvulus Wucheria bancrofti Brugia malayi Dranunculus medinensis Ancyclostoma braziliense Toxocara canis

Vector: black fly Reservoir: humans Pacific islands, Africa Southeast Asia Guinea worm Dog hookworm Dog roundworm

River blindness

Ivermectin

Anopheles, Aedes, Culex Filariasis Freshwater copepods Cutaneous Larva Migrans Visceral Larva Migrans

Diethylcarbamazine (DEC) Pull from skin thru stick Thia/Albendazole DEC/Albendazole

PLATYHELMINTHS (FLATWORMS) CESTODES (Tapeworms) Taenia solium Taenia saginata Diphyllobothrium latum Hymenolepsis nana Echinococcus granulosus Spirometra spp.

Pork tapeworm Beef tapeworm Fish tapeworm Dwarf tapeworm Hyatid disease

Scolex: head with hooks Head with suckers Causes B12 deficiency aka Extra intestinal tapeworm

Praziquantel

Surgical removal of cyst

TREMATODES (Flukes) Schistosome (blood fluke)  S. japonicum  S. mansoni  S. haematobium Paragonimus westermani Fasciolopsis buski Fasciola hepatica Fasciola gigantica

Oriental Mansons Egyptian hematuria Lung fluke Intestinal Fluke temperate liver fluke tropical liver fluke

Host: Oncomelania hupensis quadrasi snails Katayama fever (acute) Hosts: snails, mountain crab Bamboo & water chestnuts Snails and aquatic plants

Praziquantel

Bithionol

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ANTHELMINTHICS CHEMOTHERAPY FOR NEMATODES Albendazole Mebendazole (Antiox ®) Pyrantel Pamoate Piperazine Ivermectin Thiabendazole Diethylcarbamazine

Inhibits microtubule synthesis Inhibits microtubule synthesis Depletes glucose Depolarizing neuromuscular agent (nicotinic receptors) Flaccid paralysis of helminth (Blocks response of helminth muscle to Ach) Targets GABA receptors  paralysis Also affects microtubular aggregation Unknown CHEMOTHERAPY FOR TREMATODES

Praziquantel

Increases membrane permeability to calcium  contraction  vacuolization  parasitedeath CHEMOTHERAPY FOR CESTODES

Niclosamide

Inhibits oxidative phosphorylation in mitochondria of cestodes

Ascaris, enterobius, and trichuris infections Ascaris, hookworm and enterobius infestations Ascaris and pinworm Onchocercavolvulus (river blindness) Strongyloidiasis (threadworm) Filariasis

For intestinal cestodes only: T. saginata, D latum, H. nana

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PROTOZOA Protozoa Entamoeba histolytica

Disease Amebic colitis

Transmission Fecal-oral

Trichomonas vaginalis

Trichomoniasis

Sexual

Blastocystis hominis

Blastocystosis

Fecal-oral

Giardia lamblia

Leningrad’s curse Beaver fever Balantidiasis

Fecal-oral

Toxoplasma gondii

Toxoplasmosis

Isospora belli

Isosporiasis

Cat feces (oocyst) Fecal-oral

Balantidium coli

Misc Ingest RBC, Cyst has 1-4 nuclei Cigar/sausage chromatoid bodies Burning urination Yellow green frothy v. discharge

Treatment

Metronidazole

Trophozoite: “old man’s eyeglass” Metronidazole Tetracycline

Pig feces

(largest protozoan parasite, only ciliate known to cause disease)

Diagnosis: Serology, Radiology, Retina exam

Na stibogluconate and n – methyl-glucamine antimonite

Leishmanias L. tropica(Oriental sore) L. braziliensis,(Espundia) L. donovani (Kala-azar)

T. brucei gambiense T. brucei rhodesiense Trypanosoma cruzi Plasmodium P. falciparum P. vivax P.ovale P. malariae

Leishmaniasis

Sand flies

West African SS East African SS American SS Chagas’ disease

Tse tse fly

Stibogluconate Pentamidine Nifurtimox and Benznidazole

Triatomine/ Kissing bug Anopheles mosquito

Malaria

Fansidar Pyri + Sulfadoxime Cotrimoxazole

Intermediate host: Human Schizogony (asexual) Definitive host: Anopheles Sporogony (sexual)

Chloroquine is the tx of choice

ANTIPROTOZOANS o

Have one common structural feature – a quinoline ring; none except the cinchona alkaloids has a quinuclidine ring

CINCHONA ALKALOIDS

Quinine

7-CHLORO-4AMINOQUINOLINES

Chloroquine Amiodaquine

8-AMINOQUINOLINES

Primaquine

9-AMINOACRIDINES

Quinacrine

Mefloquine

Reserved for malarial strains resistant to other agents AE: Cinchonism, Abortifacient DOC for erythrocytic falciparum malaria Anti-inflammatory (use in RA and discoid lupus erythematosus) P. falciparum (curative) and Plasmodium vivax Highly supressive in P. vivax and P. falciparum Only for exoerythrocytic stages of malaria Only agent that can lead to radical cures of the P. vivax & ovale Gametocidal for all 4 plasmodia species Primarily used in the treatment of giardiasis, but is also effective against tapeworm and malaria, and topically, against leishmaniasis Should not be given with primaquine because of inc. toxicity For multi-drug resistant forms of Plasmodium falciparum Kathreen Mae Doria Cascabel

VIROLOGY DNA VIRUSES  Double stranded  Icosahedral  Replicate in nucleus

Herpes

Hepadna

Name Herpes HSV-1 Viridae HSV-2 Varicella Zoster Virus (VZV) Cytomegalovirus (CMV)

Epstein-Barr Virus (EBV)

Pox Papova Adeno Parvo

Adeno

Papova

Parvo

Clinical Gingivostomatitis (cold sores) Herpetic keratitis of the eye Encephalitis Genital herpes Neonatalherpes Varicella/Chickenpox Mononucleosis Pneumonitis (marrow patients) Retinitis (AIDS patients) Infectious mononucleosis Burkitts lymphoma Kissing virus Smallpox/Variola Molluscum contagiosum Human Papilloma Virus – warts Childhood URTI 5th Disease Erythema infectiosum “Slap cheek rash” Transient aplastic anemia crisis

Pox

Treatment Acyclovir

Acyclovir Acyclovir Ganciclovir

Supportive

Vaccine Wart removal Vaccine (Gardasil®) Self limiting Self limiting

RETROVIRUS, HIV, AIDS (RNA VIRUS)  Diagnosis: o ELISA (screening) o Western Blot Test (confirmatory)  Prevention: Education, blood screening, vaccine development  Limit growth: HAART (Highly Active Retroviral Therapy)  Treatment of opportunistic infection: o Pneumocystis carinii - Trimethoprim + Sulfamethoxazole o Toxoplasma - Pyrimethamine/Sulfadiazine o Mycobacterium - Azithromycin & Clarithromycin o Herpes, Varicella zoster - Acyclovir

Kathreen Mae Doria Cascabel

RNA VIRUSES  Single stranded  Enveloped  Helical  Replicate in cytoplasm

Hepatitis Viridae TAE (fecal-oral) Acute – caused byall

Chronic– causedby bloodborne

Orthomyxo & Paramyxo

except except except except

Reoviridae Picorna, Calilci, Reo, T cell leukemia virus Picorna, Calici, Reo, Toga, Flavi, Rhabdo (bullet) Retro & Orthomyxoviridae

Name Hepatitis A (Picornaviridae) NEVER BECOMES CHRONIC

Clinical Acute viral hepatitis Diagnosis: Serology

Treatment Immune globulin Supportive HAV Vaccine

Complications: Hepatocellular CA Cirrhosis

HB vaccine Alpha interferon Lamivudine

Anti HAV IgA – early Anti HAV IgG – late

Hepatitis B (Hepadnaviridae) ONLY DNA HEPA VIRUS HBsAg – immune HBeAg – low infectivity HBcAg – never protective

Infectious ‘Dane particle’ Hepatitis C (Flavi) Hepatitis D (incompleteRNAv)

Coinfection with HBV

Hepatitis E (Calici) Hepatitis G (Flavivirus) Influenza

Hepatitis epidemic in Asia No liver disease Flu

Type A: human and animal Type B and C: human

Respiratory Synctial Virus (RSV) Mumps Measles(Rubeola) Togaviridae (Rubivirus) Flaviviridae

Rhabdoviridae – bullet shaped Poliovirus - infect Peyer’s patches of the intestine and motor neurons

Arboviruses (Toga, Flavi, Bunyaviridae/hantavirus) Picornaviridae (Entero: polio, coxsackie A&B, echo, HAV) Rhinovirus, Coronavirus Rotavirus (1° cause of infantile diarrhea), Calici, Norwalk

Vaccine

Complication: Reyes Syndrome

A: Amantadine & Rimantadine B and A: Sanamivir & Olsetamivir

URTI, Croup Viral pneumonia Most common cause of pneumonia in infants

Supportive

Virulence: Hemagglutinin (HA) and Neuraminidase (NA)

Parainfluenza

Alpha interferon Ribavirin Control of HBV Infection

Prodome, fever, hacking cough, Koplik’s spot Rubella (German measles) Dengue/Breakbone/H fever, Yellow fever, Hepa C West Nile , Chikugunya fever Rabies Mild illness Aseptic meningitis Paralytic poliomyelitis

Ribavirin MMR Vaccine MMR Vaccine MMR Vaccine Mosquito control

Vaccine Salk – Inactivated, formalin-killed Sabin – oral, attenuated

Myo/pericarditis, meningitis Common colds, SARS, MERS Diarrhea Kathreen Mae Doria Cascabel

Herpes HIV (Antiretroviral)

Influenza

ANTIVIRAL AGENTS DNA PolymeraseInhibitors Acyclovir, Valacyclovir, Ganciclovir NRTI Zidovudine, Abacavir, Dideoxynosine, Lamivudine, Stavudine, Tenofovir, Disoproxil, Fumarase, Zalcitabine NNRTI Delavirdine, Efavirenz, Nevaripine Protease Inhibitors Amprenavir, Indinavir, Lopinavir, Nalfinavir, Ritonavir, Saquinavir Amantadine, Rimantadine, Olsetamivir

Kathreen Mae Doria Cascabel

PUBLIC HEALTH Prions - Proteinaceous Infectious Particles Transmissible spongiform encephalopathies (TSE) - fatal neurodegenerative disorders of humans and other animals

Best known animal diseases – Scrapie: sheep & goats – Bovine spongiform encepalopathy (BSE or “mad cow disease”): cattle 4 human prion diseases • • • •

CJD Kuru GSS FFI

Characteristics of prion diseases • Long incubation time • Gradual increase in severity leading to death within months of onset • No host immuneresponse • Non-inflammatory process in the brain • Neuropathological findings: – Macroscopic: normal – Microscopic: spongiform changes, neural loss and amyloid plaques w/ accumulation of PrP The infectious particles are relatively resistant to heat and many commonly used chemical disinfectants as well as irradiation BSE epidemic • *Attributed to the practice of feeding cattle (contaminated) sheep offal carnivores • Transmission from mad cows to humans  vCJD Kuru epidemic • Fore tribe, Papua New Guinea • Ritualistic cannibalism • sickness Iatrogenic CJD • Contaminated (neuro) surgical instruments, dural & corneal grafts • Cadaveric pituitary hormones

Kathreen Mae Doria Cascabel

Antibioticdarwinism Factors associated w/ resistance  Widespread, inapp. use of broad spec antibiotics  Use of antibiotics in animal husbandry & fisheries  Xs use of antimicrobial prep in soaps & cleaning sol’ns in non-healthcare facilities  Inc immunocompromised px prolonged course  Prolonged survival of debilitated px  International travel  Poverty Genetic variability • Point mutations – Aka microevolutionary change – Mutations may alter the target site of antimicrobial agent activity • Macroevolutionary change – Rearrangement* of large segments of DNA as a single event • Acquisition of foreign DNA – Carried by plasmids, bacteriophage or transposable genetic matl’s adapt antimicrobial activity Mechanism of antimicrobial resistance 1. Enzymaticinhibition 2. Alteration of bacterial membranes 3. Promotion of antibiotic efflux 4. Alterations of bacterial protein targets 5. Bypass of antibiotic inhibition Decreasing antimicrobial resistance • Withhold antibiotics • Narrowest spectrum antimicrobial agents – Staph infx: Pens vs Fluoroquinolone • Base decisions about broadness of empiric antibiotic coverage on the severity of illness • Careful hygiene, handwashing • Education

Sporadic Endemic Epidemic Outbreak Pandemic

- occasionally - constantly present in a population - many people in a given area, relatively short time - sudden - worldwide epidemic

Kathreen Mae Doria Cascabel

IMMUNOLOGY & SEROLOGY Antigen  Foreign substance, usually a protein  Part of bacteria, virus, protozoa, fungi, and endogenous (self) cells that have become altered Antibody  Immunoglobulin molecule produced by specialized B cells  Produced in response to a specific antigen  Unique, specific and form a “complex” with “their” antigen, with the intent to inactivate the antigen Antibodies are also known as Immunoglobulins, abbreviated IgG, IgM, IgE, IgA, and IgD. The immune system  Cells: o Lymphocytes and Monocytes (WBCs circulating in the blood) o Macrophages (which leave the bloodstream and migrate in the tissues)  Tissues: Bone marrow and the sub-mucosa of GIT  Organs: Spleen, Thymus, Liver (fetal), Lymph nodes Immunity  Innateimmunity o Generalized (protects the body against all invaders) o Skin and mucous membranes physical barriers o Neutrophils and monocytes (both types of WBCs), and tissue macrophages (WBCs which have migrated to the tissue  engulf all invaders (non-specific phagocytosis) o Colostrum - preformed antibodies from the mother o Protective secretions from the body such as mucus o Non-pathological bacteria that normally live in the body  inhibit the growth of pathogens 

Acquired immunity - Specific (reacts against specific antigens) o Humoral immunity  mediated by B cells (lymphocytes that mature in the bone marrow) which are spread primarily to lymph nodes and the spleen  “B” cells produce plasma cells (response to antigen) specific antibodies or Ig  Some “B” cells  memory cells  Ig IgG IgM IgA IgE IgD o

Most common, smallest (crosses placenta) First responder, Second most common Secretory Ab (mucosal surfaces) Attach to mast cells and basophils  release of histamine Growth and differentiation

Cellular immunity  Mediated by T cells  T-lymphocytes  “activated T cells” circulate throughout the body often for months  The activated cells perform much like antibodies, by binding to specific antigens Kathreen Mae Doria Cascabel

Interactions between different parts of the Immune System Tissue macrophages o Inactivate antigen  circulation  lymph node and “present” it to memory cells which reside there Helper T cells (of cell mediated immunity) aka CD4 o Primary regulators of the immune system o Produce various substances including interleukins, which are essential in fighting viral infections Complement (also known as the complement system) o Series of enzymes that are found in the blood o Assist the immune system by combining with the antigen and antibody complex causing phagocytosis of bacteria by WBCs Hypersensitivity Type I: Immediate Type II: Cytotoxic Type III: Immune Complex Type IV: Delayed

Mediators IgE antibodies Complement system Antigen and antibody complexes T lymphocytes 24 hours or more after exposure

Example Skin irritation to fatal anaphylactic shock Blood transfusion reaction Glomerulonephritis and RA Organ transplant rejection and tuberculosis testing (Mantox test)

Publichealth o Science of protecting and improving the health of communities through education, promotion of healthy lifestyles, and research for disease and injury prevention Core Areas 1. Behavioral Science or Health Education 2. Biostatistics  Identify health trends that lead to life-saving measures through the application of statistics  Often utilized in tandem with epidemiology. 3. Emergency Medical System 4. Environmental Health 5. Epidemiology  Spot and understand the demographic and social trends that influence disease and injury and evaluate new treatments. 6. Health Services Administration/Management 7. International / Global Health 8. Maternal and Child Health 9. Nutrition 10.Public Health Laboratory Practice 11.Public healthpolicy  Legislative action at the local, state, and federal levels. 12. Public healthpractice Types of Immunity 1. Natural (Innate) Immunity 2. Acquired Immunity Active Immunity a. Naturally Acquired Active b. Artificially Acquired Active Passive Immunity a. Naturally Acquired Passive K a t h b. r e eArtificially n M a e Acquired D o r i a CPassive ascabel

Ottawa Charter for Health Promotion  First International Conference on Health Promotion  Ottawa, 21 November1986  Health Promotion – process of enabling people to increase control over, and to improve, their health.

PHARMACY EDUCATION SUFFERED FROM… (Gibson, 1972) o The lack of definition of PH in Pharmacy o The lack of perceived relevance to pharmacy students o The lack of a textbook focusing on the roles of pharmacists in PH o The lack of pharmacy faculty educated in and with appropriate experience to teach PH o The lack of sites where students could become involved with PH projects and work with PH personnel Millenium Development Goals

Philippine health caresector Institutions • Department of Health - Its mission is to guarantee equitable, sustainable and quality health for all Filipinos, especially the poor, and to lead the quest for excellence in health. • Bureau of Food and Drugs (BFAD) - Its main responsibility is to license and regulate the delivery of pharmaceuticals in the Phil - It is also tasked to test the safety of food and cosmetics. • Philippine Health Insurance Corporation (RA 7875) - Promoted the membership of every Filipino in the healthcare program, particularly the indigent sectors of the population. Consequently, it established PhilHealth Public & Privatehospitals Industry Associations Kathreen Mae Doria Cascabel

Some healthprograms/Campaigns Fourmula One (F1)  launched as a blueprint of reform implementation aiming for a more responsive health system, more equitable health financing, and better health outcomes.  Within the medium term 2005-2010, F1 was devised to address the issues on fragmentation of the Philippine health system and inequity in health care.  Also, F1 intended to achieve speedy, precise, and well-coordinated critical reforms to improve the health system of the country. Formula One (F1) Components: 1. Health Financing 2. Health Regulation 3. Health Service Delivery 4. Good Governannce in Health Philhealth Sabado - "PhilHealth Sabado. Magseguro. Magparehistro“ DOTS •

Elements o Political commitment with increased and sustained financing o Case detection o Standardized treatment, with supervision and patient support o An effective drug supply and management system o Monitoring and evaluation system, and impact measurement

Yosi Kadiri  2009 Global Adult Tobacco Survey  started in the early 90’s during the term of former undersecretary Juan Flavier  R.A. No. 9211, known as the Tobacco regulation Act of 2003  revived

4 o-clockHabit  First launched in 1996 which encouraged communities to seek and destroy breeding places of mosquitoes every 4pm daily  “Stop, Look, and Listen”  Aksyon Barangay Kontra Dengue – DOH + DILG Alcoholics Anonymous (AA)  Fellowship of men and women who share their experience, strength and hope with each other that they may solve their common problem and help others recover from alcoholism

Kathreen Mae Doria Cascabel

QUALITATIVE-QUANTITATIVE CHEMSTRY   

Aka If a sample is a drug: Branch of chemistry which provides information relative to the composition of matter

Divisions of Analytical Chemistry 1. Qualitative - composition determination 2. Quantitative - determination of the proportion of components Classification of Analysis 1. Based on Sample Size a. Ultra micro b. Micro c. Semi micro d. Macro

: <1 mg : 1-10 mg : 10-100mg : 100mg-1g

2. Based on extent of determination a. Proximate b. Ultimate 3. Based on nature of methods a. Classical aka General/Chemical b. Instrumentation - based on specific physical or chemical properties of the analyte c. Miscellaneous/Specific method - involves crude drugs and natural products derived from them 1. Based on materials used a. Chemical b. Physical c. Biological Basic Principles of Analysis A. pH   

Number of gram equivalent of H per liter of solution pH = -log[H] or pH = log 1/[H] Protolysis – transferring of proton from one molecule to another Autoprotolysis – transferring of proton from one molecule to another identical molecule Amphoteric – property where a substance can act either as acid or base Henderson-Hasselbalch Equation Weak acids

�𝐻= �𝐾�+ 𝑙�𝑔 ��𝑙� ��𝑖 �

Weak bases

�𝐻 = �𝐾𝑤 − �𝐾� + 𝑙�𝑔 ����

��𝑙 �

Kathreen Mae Doria Cascabel

ProblemSolving: 1. Calculate the pH of a solution which contains 3.7 x 10-2 mole of HAc and 4.8 x 10-2 mole pf Na Ac in a liter of solution pKa 0.26

2. A buffer solution containing 0.1M each of HAc and NaAc, is present with Ka of HAc = 1.8 x 10-5 at 25°C. Find the pH of the solution.

3. What is the ratio of the salt and acid conc if the solution has a pH of 4.5 and pKa of 4.76?

B. Buffer Capacity  Ability of a buffer solution to resist changes in pH upon addition of acid/alkali  Van slyke - Responsible for a quantitative expression - Amount in g/L of strong acid or a strong base required to be added to a solution to change its pH by 1 unit - Higher buffer capacity, lower change in pH  𝜷𝒎𝒂𝒙 = �. ��� 𝑪

CLASSFICATION USED FOR OFFICIAL ASSAY a. Titrimetry b. Gravimetric methods c. Spectrometric methods d. Electrometric methods e. Chromatographic methods f. Miscellaneous methods Kathreen Mae Doria Cascabel

TITRATION aka Volumetric Analysis -

Stoichiometric Point/Equivalence Point/Theoretical Endpoint (before the endpoint) Endpoint (experimental; indicates end of titration)

Reactants: 1. Titrant/Standard Solution 2. Titrand/Analyte 3. Indicator

- known concentration (determined by Standardization-uses Primary std) - one being analyzed - weak acids or bases that change color in pH change Acidic Basic MethylRed Red Yellow Phenolphthalein Colorless Pink Phenol Red Yellow Red Malachite Green Yellow Green MethylOrange Pink Yellow Bromothymolblue Yellow Blue Thymol blue Yellow Blue

Types of Titration According to the Number of Titrants:  TITRANT o Direct o Indirect – has preliminary step before titration  TITRANTS o Residual or Back o First titrant is added in excess o Excess of the first titrant is titrated with another titrant o Why? Slow, Sharp endpoint, Sample insoluble, Volatile FORMULA � % 𝑷 = �

𝒙 � 𝒙 𝒎𝑬 � 𝒆 ����

𝒙 ���

Ex. A 0.30g sample of the NaHCO3 (MW = 84 g/mol) requires 4 mL of a 0.975 N HCl. What is the % purity?

%𝑷 = [(��)� –(��)�] 𝒙𝒎𝑬� 𝒙 � 𝒆 ��� ���� Ex. A 0.2185g sample of NaCl was assayed using Volhard method using 50ml of 0.0998 N silver nitrate and 11.9ml of 0.1350 N ammonium thiocyanate. Calculate % NaCl (MW = 58.45).

Kathreen Mae Doria Cascabel

Volumetric Solution (VS) 1. Primary standard 2. Secondary standard Titer – grams of a substance that is chemically equivalent to 1 ml of a volumetric solution Buret used in titration: Mohr : rubber tube + pinchcock Geissler: glass stopper Cleaning solutions of buret and glasswares: 1. Sodium dichromate in sulfuric acid (carcinogenic) 2. Ammonium peroxydisulfate in sulfuric acid 3. Trisodium phosphate solution 4. Synthetic detergents BLANK DETERMINATION – perform the entire titration without the analyte; check for possible sources of error

Types of Titration Based on Chemical Reaction Neutralization Acidimetry Alkalimetry

I.

Redox Permanganometry Cerimetry Iodi & Iodometry Volumetric Precipitation

NEUTRALIZATION a. Based on Media Used  Aqueous titration SA SB + WA SB + SA WB + WA WB +  Non-aqueous titration WB + salt Relatively SB WA SA

Ion Combination Complexiometry Volumetric precipitation

: Phenolphthalein, methyl red, methyl orange : Phenolphthalein : Methyl red : No indicator : C. violet, methylrosaniline Cl, quinaldine red, m. green : Methyl red, methyl orange : Azo-violet, o-nitro-aniline (very WA) : Thymol blue

b. Based on Titrant  Acidimetry  Alkalimetry

Kathreen Mae Doria Cascabel

ACIDIMETRY

Aqueous VS

ALKALIMETRY

Nonaqueous

H2SO4, HCl

1 std Indicator mo, mr,phen

Aqueous

Nonaqueous

Perchloric acid

NaOH, KOH

Na/Li methoxide

K bipthalate

K bipthalate

Benzoic acid

mo, mr, phen

Azo-violet

HCl, H3PO4, Boric acid

Phenytoin

Crystal violet Malachite gr ee n

Direct Residual II.

Caffeine, NaOH, NaCO3, NaHCO3, Na salicylate MOM, Zno, NaK tartrate

Metacholine

ASA

REDUCTION-OXIDATION

Iodometry (Indirect) Na thiosulfate/K arsenite K dichromate

VS

Permanganometry KMnO4

Cerimetry Cerric sulfate

Iodimetry (Direct) Iodine

1 std

Na oxalate

As2O3

As2O3/Na thiosulfate

Indicator

Orthophenanthroline Menadione FeSO4

Starch

Direct

KMnO4 H2O2 TiO2

Indirect

Cherry Juice (Malic acid)

Reducing Agents:  Ascorbic acid  Na metabisulfite  Na thioglycollate  Iodine tincture RA + I2  Iodide

Starch Oxidizing agents: Ferric/cupric salt  Chlorine (NaOCl)  Phenol Resorcinol OA + excess KI  Iodine

Residual

Na and K nitrite R B

Blue

Blue disappears

Endpoint Purple

* Diazotization Assay (direct titration with Na nitrite) – Sulfa drugs III.

COMBINATION OF IONS

Method Volhard (residual) Mohr Gay-Lussac Liebig

Fajans

Manifestation Colored ppt 2nd ppt Cessation of precipitation Turbidity

Change in color of ppt

VOLUMETRIC PRECIPITATION VS Indicators Ammonium Ferric ammonium sulfate thiocyanate Silver nitrate Potassium chromate

Na tetraphenylboron

1 std Silver nitrate NaCl NaCl

Bromophenol blue

K biphthalate

Dichlorofluorescin (DCF), Eosin Y, Tetrabromophenolphthalein Ethyl Ester (TEE)

NaCl

Example Aminophylline, NaCl SLS NaCl Organic N compounds Phemylephrine Tubocurarine

Kathreen Mae Doria Cascabel

COMPLEXIOMETRY Ligand - molecules which provide a group of attachment/binds to metal ion Multidentate ligand - more than 1 group that binds the metal ion; EDTA is haxadentate - equilibrium constant for the reaction of a metal ion with a ligand Formation constant Auxiliary complexing agent o Binds to metal strong enough to prevent hydroxide from precipitating but weak enough to give up the metal ion when EDTA is added o Used to permit metals to be titrated in alkaline solutions for EDTA Masking agents - to determine conc of a metal in presence of another metal Demasking agents - releases the metal ion from the masking agent VS 1 std Direct Residual Indicators

Disodium EDTA Ca carbonate Ca carbonate, Ca gluconate, ZnO Alum, Aluminum Hydroxide, Bi Metal-ion indicators: Calmagite Hydroxynaphthol blue Eriochrome black Murexide Pyrocatechol violet

Masking agents and ElementsMasked Triethanolamine Thioglycol Cyanide Fluoride Dimercaprol

Al, Fe, Mn Hg, Cu, Bi Zn, Co, Ni, Cu Ca, Mg, Al

GRAVIMETRY  

Separate the constituent from the sample and then weighing the product Determining the weight of a substance in a sample from the weight of a chemically equivalent amount of some other substance o Mg citrate oral sol’n for MgO (precipitant: Oxine) o Na2SO4 in SLS o NaCl (using 55 AgNO3) o Sodium HPO4 o Sulfurated potash (precipitated as CuO) o Mercury (precipitant: H2S)

Loss on ignition - % test material volatilized and driven off when substance is ignited (bright red heat) Loss on drying - amount of volatile material driven off (110-120°C) Very dull red heat 500 – 550°C Dull red heat 550 – 700°C Bright red heat 800 – 1000°C Yellow red heat 1000 – 1200°C White heat 1200 – 1600°C 𝑮� %𝑷= ��� 𝒙 𝑮� 𝒙 ��� ��� Ex. If a 0.46 g sample of KI yielded 0.7215 g AgI precipitate by gravimetric assay. What would be %P of KI?

Kathreen Mae Doria Cascabel

Physico-Chemical Methods of Analysis

UV Vis Near IR Medium IR

SPECTROMETRY o o

Far IR Amount of radiant energy that has interacted with the chemical species Power of transmitted, fluorescent, reflected or emitted light Chromophore - functional groups that interacts with the radiant energy Electromagnetic spectrum - system of energy propagated in a wave form - number of complete cycles that pass a single point per second Frequency - length of one complete wave or cycle Wavelength - a device with a light source, prism, filter and a sensor of energy Spectrophotometer

200-380 nm 380-780 nm 780-3000 nm 3-15 um 3-8 (group freq.) 8-15 (fingerprint) 15-300 um

Spectrophotometry o o

Measurement of radiant energy absorbed or transmitted at a definite and narrow wavelength Concentrated = ↑absorbance ↓transmittance (opposite with diluted) Beer’s Law Lambert’s/Bouger’s Law Beer-Lambert’s/Beer-Bouger’s

Method Flame Spectroscopy  FES (Flame Emission)  AAS (Atomic Absorption) Fluorometry Turbidimetry Nephelometry Colorimetry (no prism used) NMR (Nuclear Magnetic Resonance) MS (Mass Spectroscopy) Infrared Spectroscopy

- transmittance decreases when concentration increases - transmittance decreases when thickness of the solution increases - absorbance is directly proportional to the path length and concentration

Principle

Notes Elemental analysis

Same with flame test

Measures fluorescence Transmitted light Reflected light Vis spectrum Magnets Bombardment of electrons result to fragmentation Absorption of energy in the IR region is associated with vibrational transitions of atoms w/in molecules

Vit. B1, B2, Phosphorous Antibiotics, B5, B12 Colored substances 3D structure of molecules Structure, MW Structure determination

ELECTROMETRIC METHODS 1. Voltammetry  Info about the analyte is derived from the measurement of a current as a function of the applied potential to a tiny micro electrode Polarography – method of analysis based on current measurement resulting from analysis of an electronegative species at a given electrode potential under controlled conditions 2. Potentiometry  Measurement of cell voltage in order to extract chemical information  Measurement in ion activity, in very diluted solution  Branch of electrochemistry which deals with the study of measurement of electrical potential  For pH determination (uses glass electrode) o Indicating electrode- develops potential which varies with conc or activity of ions o Reference electrode- maintains a constant potential Kathreen Mae Doria Cascabel

CHROMATOGRAPHY o o o o

Used to separate and identify the components of a mixture based of the differential affinities of the solutes between 2 phases Used to separate colored and colorless compounds Development – separation of the sample mixture into a series of narrow bands 2 Phases: 1. Stationaryphase - fixed bed core of large surface area 2. Mobile phase - moves through or over the surface of the stationary phase

Method Adsorption liquid-solid chromatography (LSC) Column

Description Mobile phase compete with the analyte for polar adsorption site Simplest type consisting of a suction flask and cylinder tube constricted on end

TLC

Spotting of a sample followed by passage of solvent through the adsorbent to separate components Difference in partition coefficients of substances between two immiscible liquids

Partition Chromatography Ex. Paper Chrom

Reversed-Phased Ion-Exchange

Retention factor (Rf) = distance solute/distance solvent Partition phenomenon Partition phenomenon

Molecular Exclusion/Gel Differential migration of Filtration/Gel Permeation solute molecules is based on molecular size Gas Analysis of volatile compounds HPLC Most widely used Fast, accurate, precise Supercritical Liquid Chromatograpy – for very volatile compounds

Stationary Solid

Mobile Liquid/Gas

Solid (adsorbent)  Purified siliceous earth  Activated alumina  Silica gel  Calcium carbonate Silica gel G

Liquid

Water in filter paper (adsorbent, fixed porous material)

Organic solvent

Non-polar solvent Coated solid referred to as resin Gel

Polar solvent Liquid

Solid or a non-volatile liquid

Gas

Solid

Kathreen Mae Doria Cascabel

MiscellaneousMethods

GASOMETRIC ASSAY o

Measure of gas liberated or removed under the conditions described in the assay

TEST FOR VOLATILE OILS 1. Specific Gravity  Westphal balance  Pycnometer method at 25°C  Range: 2. Polarization  Power of rotating the plane in polarized light (optical activity)  Polarimeter measures optical activity  Source: Iceland spar  Prism: Nicol 3. Refractive Index  Ability to bend light – bending of light passing from one medium to another  Refraction Index of refraction – ratio of velocity of light in air to velocity of light in medium 4. Specific assay  Ester value  Amount of alcohol Transform free alcohols in corresponding acetate by boiling with acetic anhydride in acetylization flask then determine saponification value

5. Determination of aldehydes and ketones  Bisulfite Method (using cassia flask)  Hydroxylamine Method/Titrimetric

- form water soluble addition product - hydroxylamine is made to liberate HCl

6. Assay of phenol content  With NaOH (phenol constituents – soluble in alkali)

FATS AND FIXED OILS # of mg of KOH requiredto Acid value Estervalue Saponification value/Koettsdorfer# Hydroxyl value Iodine value

Formula

Neutralize the free acids in 1g Saponify the esters on 1 g Neutralize free fatty acids and saponify esters

AV/EV = N x V x56.11 Wt sample SV = AV + EV

# of KOH equivalent to OH content of 1 g subs # of mg of Iodine absorbed by 100 g substance Measures degree of unsaturation

Same as AV IV = NV (0.1269) x100 Wt

Hanus – I2 & Br2 in glacial acetic acid Wijs – I2 monochloride in glacial acetic acid Hubl’s – unofficial

Drying: >120 Semi-drying: 100-120 Non-drying: <100

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ASH CONTENT DETERMINATION o o

Residue remaining after incineration Represents inorganic salts (phosphates, silicates, sulfates, carbonates) – impurities 1. Total Ash 2. Acid Insoluble Ash(AIA) % AIA = wt of AIA x 100 wt total 3. Water-solubleAsh 4. Sulfurated Ash

- no treatment - part of total ash not soluble in dilute HCl (determines silicates)

- represents soluble chlorides, alkali, NH4 salts (T: 450°C) - treated with sulfuric acid

WATER CONTENT DETERMINATION MethodI

MethodII

MethodIII

Karl Fischer Method/Titrimetric KFR = 1 ml is equivalent to 5mg of water Composition:  Anhydrous methanol – prevents pyridine-sulfur complex  Iodine – reacts with water to hydroiodic acid  Sulfur dioxide – reacts with water to sulfur trioxide  Pyridine – prevents reversal of action Azeotropic Distillation/ Xylene/ Toluene Method Solvent: xylene, toluene Also used in Alcohol Content Determination Gravimetric For:  1-2 g chemical sample  Vegetable drugs  Biologics  Crude drugs

% water = ml KFR x EF x 100 wt

% water = ml reading x 100 wt

ALCOHOL CONTENT DETERMINATION Method I: Distillation Method  High alcohol content: low specific gravity Method II: Gas Liquid Chromatography  Std soln : Absolute alcool in water  Internal std : Acetonitrile in water  Carrier gas : He or N  Detector : Flame ionization detector

NITROGEN DETERMINATION (Kjeldahl Method) BIOLOGICAL TESTING o

Drug of natural origin a. Animal assay b. Microbial assay c. Pyrogen tesr d. BET (Bacterial Endotoxin Test) Kathreen Mae Doria Cascabel

QUALITY ASSURANCE-QUALITYCONTROL Quality

-

Totality of characteristics or features of a product that bears its capacity to satisfy stated or implied needs Determines degree of acceptability

Quality Assurance

Sum total of the organized arrangements made with the object of ensuring that products will be consistently of the quality required by their intended use QA DepartmentalFunctions  Assures policies are followed inept to economic issues associated with manufacturing and distribution of product  Cooperate with regulatory agencies and final authority for product acceptance or rejection  Helps to identify and prepare necessary SOP’s relative to control of quality  Audit and quality monitoring

Quality Control

Part of GMP concerned with sampling, specifications, testing, organization, documentation and release procedures QC Functions  Testing and acceptance of only highly quality raw material, representative samples  IP tests against criteria  Monitors environmental conditions  Control packaging components

3 Main Areas of QC 1. Raw Material Quality Control (RMQC) 2. In Process Quality Control (IPQC) 3. Finished Product Quality Control (FPQC) Monograph

Document that specifies all the tests to be conducted on a product and/or appropriate references containing details of procedure and expected result

Certificate of Analysis

Document with the results of all tests conducted on material to show compliance or noncompliance with the standard specifications

Formula

This is concise and precise statement of the ingredients that comprise the product, together with the percentage and/or weight of each.

Raw material specification

This should enumerate the characteristics of all the materials that go into the product and the permissible range of purity of each ingredient.

StandardOperating Procedure Finished product specification

This is a step by step method on how to go about a job.

Sample

Finite number of objects selected from a batch

This should cover all characteristics that affect the proper performance, purity, safety and stability of the product.

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DEFECTS - undesirable characteristic of a product, failure to conform to specifications. According to measurability Variable defect - can be measured directly by instruments Attribute effect - cannot be measured directly by instruments According to seriousness or gravity - may endanger life or property and may render the product non-functional. Critical defect (Absence of warning in a label for a potent drug) Major defect - render the product useless (crack in a bottle) - does not endanger life or property nor will it affect the function but nevertheless Minor defect remains a defect since it is outside the prescribed limits (slight deviation of the color of the label from the color standards) According to nature Ocular defect - visible (foreign particular contamination) Internal defect - not seen although present (subpotent drug product) Performance defect - defect in function (suppository that does not melt at body temp)

Sources of Variation Materials

Machines

Methods

Men

Analytical Balances Class 1.1

Example a. Variation between suppliers of same substance b. Variation between batches from same supplier c. Variation within a batch a. Variation of equipment for the same process b. Difference in adjustment of equipment c. Aging and improper care a. Inexact procedures b. Inadequate procedures c. Negligence by chance a. Improper working conditions b. Inadequate training, and understanding c. Dishonesty, fatigue and carelessness

: For low capacity (1 to 500mg), high sensitivity balances

Class 1

: High precision standards for calibration, for weighing accurately quantities below 20mg

Class 2

: May be used for qty greater than 20 mg

Class 3 & 4

: Used with moderate precision lab balances

Class 3

: For qty greater than 50mg

Class 4

: For qty greater than 100mg

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Raw Material Quality Control (RMQC) ADULTERATION  Debasement of article Sophistication “true adulteration”, Addition of inferior material Substitution Entirely different from the one requested Admixture Add through accident, ignorance, carelessness Spoilage Quality destroyed by microorganism Deterioration Quality impaired due to environment, insects, etc A. ID TEST 1. Chemical 2. Physical

- Indication: change in color, precipitation, evolution of gas Spec gravity Solubility Refractive index Optical activity Boiling point Melting point

3. Instrumental IR spec UV-vis spec HPLC NMR B. TEST FOR PURITY (POTENCY) 1. Monograph (Assay) Chemical Instrumental Biological

- wt of sample/wt of std (water) (Pycnometer and Westphal balance) Alcohols at 15.56°C, Water at 25°C - Dissolution of compound at a suitable solvent - Bending of light (Abbe refractometer) Ratio of velocity of light in air and velocity of light in a sample - Angle of optical rotation of a plane polarized light (Polarimeter) - Vapor pressure is equal to atmospheric pressure

- Vibration, compared to standard - Excitation – absorbed and transmitted light - Retention factor - Amount of magnetic signal/field resonance

- Titration - HPLC, GC

C. LIMIT TEST 1. Gross impurities (insoluble matter, dirt) 2. Biological impurities (microorganisms, cell debris, degradation products) 3. Chemical impurities (trace metals) - AgNO3  AgCl Chloride Sulphates - BaCl2  BaSO4 Arsenic - Diethylthiocarbamate(vis-spec 535-45mm) Na and Ca - Flame spectroscopy Iron - Ammonium thiocyanate  blood red complex - H2S  black Heavy metals Except:  Zinc (white)  Cadmium (yellow)  Tin (orange)  Mn (pink) Kathreen Mae Doria Cascabel

QC Procedures Solid Dosage Forms A. GRANULES ANDPOWDERS Particle Size

Sieving –particle scattering technique Mesh # - # of linear openings per sq inch

Angle of Repose

arc tan height radius Methods:  Static Angle of Repose o Fixed funnel method o Fixed bed cone o Tilting box  Kinetic Angle of Repose Weight Bulk volume

Bulk Density Tapped Density

Weight Tap volume

Carr’s Index TD – BD x 100 TD

or

Hausner’s Ratio

TD BD

Bulk V TapV

Porosity

Void x 100 *Void = Bulk – true volume x 100 Bulk volume Method 1: KFM/Titrimetric % water = ml KFR x EF x 100 wt Method 2: Azeotropic % water = ml reading x 100 wt Method 3: Gravimetric

Moisture Content

Content Uniformity

Coarse granule : retained 20 Good granules : pass 20, retained 40 Fine granules : pass 20 and 40 <25 Excellent 25-30 Good 30-40 Fair (add glidant) >40 Poor

or

Bulk V – Tap Vx 100 Bulk V

Excellent 5-13 Good 12-16 Fair to passable 18-21 Very fluid 23-28 Fluid cohesive 18-35 Extremely poor, cohesive >40 Good <1.25 Poor >1.25 Fair to Passable 1.25-1.5

0.55-2% ↑ moisture: sticking, picking

↓ moisture: capping, chipping, lamination

% active component = 98-102% active comp

Shape

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B. TABLETS Equipment Hardness

Stokes Monsanto Strong cobb Pfizer Erweka Schleuniger

- spring - air pump - pliers - susp weight - horizontal

Formula and Acceptance Criteria Plain uncoated Buccal Chewable,SL

4-10 7-10 2-3

Thickness

Micrometer caliper

± 5% of standard thickness

Friability

Roche Friabilator  100 rev (25 rpm, 4 mins)  >650 mg (10 tab) <650 mg (20 tab)

% Friability = Initial – Final x 100 Initial AC = nmt 1% New = nmt 0.8%

Disintegration

Basket Rack Assembly  6 cylindrical tubes  10 mesh wire at the bottom Media temp: 37 ± 2°C

 

Dissolution

Temp: 37 ± 0.5 °C Type 1 Basket Capsules Type 2 Paddle Tablet Type 3 Reciprocating Cylinder Type 4 Flow Through Cell

Q tolerance = % dissolved accdording to LC Sample Criteria 1 6 All units should have >Q + 5 Q 90% = 95% 2 +6 Ave of 12 units nlt Q No unit less than Q-15% 3 +12 Ave of 24 units nlt Q Nmt 2 units below Q-15% No unit below Q-25%

Weight Variation For tab >50mg

Steps:    

 

Content Uniformity For tab <50mg

Weigh Average Upper and lower limits Compare

Endpoint: Impalpable core If 1 or 2 did not disintegrate, perform 2 trials more  Nmt 2 of the 18 samples tested failed to disintegrate Plain Water 30 mins EC Water 5 mins Gastric juice 1 hr Intestinaljuice 1 hr Buccal Water 4 hrs SL Water 3 mins

Nmt 2 tab differ by more than % var No tab differs by more than double %var <130 mg ±10% 130-324 mg ± 7.5% >324 mg ± 5% 1 10 2 20

85-115% LC RSD nmt 6% Nmt 1 unit is outside 75125% LC RSD nmt 78%

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Non-Sterile Oral/Topical Products Microbial Limit Test

Test Total Aerobic Count Total Yeast/Mold 1. SOLUTIONS 1. Appearance 2. Physical 3. Chemical B. SUSPENSIONS

Medium TSA (Tryptone, Soya Agar/broth) PDA (Potato Dextrose Agar)

Conditions 30-35°C (48-72 hrs) 20-25°C (5-7 days)

- visual inspection for soluble matter or impurities - odor, color, taste, pH, viscosity, clarity - degradation products, potency

- should settle | pour readily and evenly | particles remain fairly constant Formula/Description Vs = Vu (settled) Vo (unsettled)

Criteria The lower Vs, the better Vs <1: not flocculated enough Vs =1: “flocculated equilibrium”

Redispersability

Amount of force to redisperse particles (cake test)

Adv: 100% redispersible with min agitation No sediment should remain at the base after shaking

Particle Size Measurement

Microscopy (micrometer lens)

Ideal size: 2µm (suspension) Solution: <0.1 Colloidal: 0.1-1

Rheological Properties

Rheology = flow 1. Viscosity 2. Flowability

Pour out readily and evenly from container (Pseudoplastic/shearthinning)

Sedimentation Volume

Temperature & Gravitational Stress

Test for crystal growth at 40°C

Zeta Potential Determination

Repulsive forces among particles

Inc zeta potential = slower setting

C. EMULSIONS 1. Tests for Phase Inversion, Creaming, Cracking, Phase Separation O/W DyeSolubility Amaranthgreen Dilution Miscible Conductivity + UV Absorption/Fluorescence Cobalt Chloride + (blue to pink) Direction of Creaming Upward

W/O Sudan red Immiscible + Downward

2. Electrophoretic Analysis 3. Particle Size 4. Gravitational and Temp Stress Test (50-70°C) Kathreen Mae Doria Cascabel

D. MICROBIAL ASSAY FOR ANTIBIOTICS

Cylinder Plate Method (Zone of Inhibition)

Turbidimetric Method

TestOrganism S. aureus S. cerevisiae Micrococcus luteus Bordetella bronchiseptica B. subtilis S. epidermidis Mycobacterium smegmatis P.aeruginosa S. aureus

K. pneumoniae S. faecium Lactobacillus plantarum S. cerevisiae E. coli Lactobacillus leihmanii E. BIOLOGICALASSAYS Cat Chicken Dogs Pigeons Rabbit

Rat Rat (female) Rat (male) Rat (rachitic) Sheep

Antibiotic Cephalothin, Cloxacillin, Nafcillin, Pen G Amphotericin B, Nystatin Bacitracin, Erythromycin Polymyxin B Vancomycin Gentamicin, Neomycin Bleomycin Carbenicillin Amikacin, Cycloserine, Demeclocycline, Doxycycline, Kanamycin, Tetracycline, Tobramycin Neomycin, Streptomycin Gramicidin Ca pantothenate Candicidin Chloramphenicol Cyanocobalamin

Glucagon Oxytocin PTH Digitalis Tubocurarine Metocurine Insulin Corticotropin Chronic gonadotropin Vasopressin Cod liver oil Heparin Protaminesulphate

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Parenterals (Sterile) Sterility Test

Methods: Primary - membrane filtration | 7 days incubation Direct transfer - direct inoculation | 7-14 days incubation CultureMedia Tryptone Soya Broth (TSB) Fluid thioglycollate (FTG)

- Aerobes, 20-25°C - Anaerobes, 30-35°C

Biological Indicators

Test for Pyrogens

BET/LAL

Pyrogen Test

Bacteria Used

Moist heat (autoclave) Dry heat (oven) Ethylene oxide Radiation

B. stearothermophilus B. subtilis B. stearothermophilus Both + B. pumilus

(+) formation of gel/development of color in presence of material endotoxins and lysate of amebocytes of: horse shoe crab (Limulus polyphemus) o o o

(endotoxin)

Method of Sterilization

3 healthy mature rabbits 10ml/kg in ear vein Rectal temp recorded at 1,2,3 hrs after injection or 30 mins interval 1 hr after injection Stage Sample Acceptance Criteria 1 3 Nmt 0.5°C temp rise 2 +5 Nmt 3/8 temp tise 0.5°C Total temp rise nmt 3.3°C

SafetyTest

Bioassays  determine cytotoxicity of parenteral components Uses mice or guinea pig

Particulate Matter Test  Presence of particles may be harmful  emboli phlebitis

Equipments: 1. Electronic Particle Counter  Measure size, # by means of shadow cast by particles as it passes through high-intensity light beam (light obstruction method)  D/A: non-specific, cannot differentiate air bubbles from particulate matter 2. Membrane Filtration Technique  Collects particles larger than rated pore size  D/A: saturable, slow counting

Leaker’s Test

Negative pressure within incompletely sealed ampule while submerged in dye sol’n Dye: 1% Methyleneblue

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Packaging Material Quality Control (PMQC) 1. Powdered Glass Test  Performed on ground/powdered glass to expose internal surface  Tests the leaching potential of the glass  Alkali treated with 0.02 N sulphuric acid 2. Water Attack Test  Exposure of the glass with sulphur dioxide at 121°C Type 1 Type 2 Type 3 Type 4

Treated borosilicate Treated soda lime Soda lime General purpose soda lime

Parenterals Parenterals Parenterals Nonparenteral

Powdered glass Water attack Powdered glass Powdered glass

Glass Defects  Cracks – partial fractures generally found in the heel, sometimes at the shoulder  Freaks – odd shapes and conditions that render container completely unusable  Filaments – hair-like string inside the bottle BIOLOGICAL REACTIVITY TESTS  In-vitro – includes agar diffusion, implantation, elution tests  In-vivo In-vivo Test Animal Used Notes Systemic Injection Albino mice For elastomeric material esp plastics Intracutaneous Albino rabbits For elastomeric material esp plastics Eye Irritation Albino rabbits For medical devices & ophthalmic containers Implantation Rabbits For material in direct contact with tissue

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STABILITY o o

Capacity of drug to remain within specifications Minimum Acceptable Potency Level: 90% Labeled Potency

AcceptanceStability  Time in storage and use in which a particular formulation in container remains within P, C, Toxicological and Bioavailability specifications Stable Product  One that retains its requisite: o Physical properties o Chemical integrity and LP o M: sterility o T: No significant inc in toxicity o Therapeutic effect remains unchanged Expiration Date – time in which the preparation will remain stable when stored under recommended condition Harmful Events – decrease in therapeutic activity, appearance of toxic substances Significant change – change from initial content of 5% or more Errors in estimating Expiration Date: Type 1 (α error) – too early, Type 2 (β error) – too late Study

Storage Condition

Min. time period

Long-term

25°C ± 2 | 60% RH ± 5

12 months

Intermediate

30°C ± 2 | 60% RH ± 5

6 months

Accelerated

40°C ± 2 | 75% RH ± 5

6 months

Climatic Conditions Zone I - Temperate Zone II - Mediterranean/Subtropical Zone III - Hot, dry Zone IV - Humid/Tropical

Kathreen Mae Doria Cascabel