1 General Laboratory PracticesSubash Chandra Verma Assistant Research Officer Chemistry Central Council For Research In Ayurveda And Siddha, Dept. Of Ayush, Janakpuri, New Delhi

2 The World Health Organization (WHO) estimatesThe World Health Organization (WHO) estimates? 4 billion people use herbal medicine. Herbal medicine is a major component of all traditional medicine  Ayurvedic  Homeopathic  Unani  Siddha Medicine

3 Plants as source of drugsAround 9000 species of plants are used by the different systems of medicine in India System Plants Ayurveda 2351 Folk 5137 Siddha 1785 Unani 979 Homoeopathy 506

4 Export of Herbal Products From India

5 RAW MATERIALS OF DIFFERENT ORIGINUSED IN ASU Drugs Plants Metals/ Minerals/ Precious stones Marine / Animal Products

6 Plant parts used in Indian System of Medicine% Flowers 10 Stem 6 Fruit 7 Wood 3 Bark 14 Whole plant 16 Rhizome 4 Root 29 Leaves Seeds 5

7 GMP & GLP FOR ASU Drugs Manufacturing Quality ControlQuality Assurance

8 Schedule “T” under rule 157 of Drugs and Cosmetics (Amendment) , 1999Raw materials used in manufacturing of drugs are authentic, of prescribed quality and free from contamination. The manufacturing process is as has been prescribed and maintains standards of purity. Adequate quality control measures are taken and The manufactured drug which is released for sale is of acceptable quality. To achieve the objectives listed above each licensee shall evolve methodology and procedures for manufacture of drugs which should be documented as a manual and kept for reference and inspection. (Vaidyas, Sidhdhas and Hakeems dispensing medicines to their own patients are exempted)

9 GMP Norms Factory PremisesThe manufacturing plant should have adequate space for: Receiving and storing raw materials Manufacturing process areas Quality control unit including in-house testing facilities Finished goods store Office Rejected goods store (Minimum for any one system 1200 sq feet covered area with separate cabins for each activity. Additional 400 sq feet for each additional system)

10 Location and SurroundingsGeneral Requirements Location and Surroundings Away from open sewage, drain, public lavatory and factories producing obnoxious odor, fumes, dust or smoke. Buildings Hygienic and free from cobwebs, insects and rodents Designed to prevent Cross-contamination Water Supply Pure and Potable quality of water. Adequate provision for washing of premises. Disposal of Waste Predisposal Treatment and guidelines for pollution control to be followed.

11 Health, Clothing, Sanitation and Hygiene of WorkersWorkers to be free from contagious diseases Uniform Suitable for climate and nature of work including coverings for hands, feet and head wherever required Facilities for personal cleanliness Clean towels, soap, scrubbing brushes, lavatories, change rooms & place for keeping personal belongings. Medical Services First aid facilities should be available. Medical Examination: At the time of employment and at least once in a year Free from infectious diseases.

12 Raw Materials Store Separate and adequate facilities forPrevent raw material contamination or rodents & insect infestation. Preserve self life Raw materials of metallic origin Raw materials of mineral origin Raw materials from animal source Fresh herbs Dry herbs or plant parts Excipient etc. Volatile oils, perfumes and flavors Plant extracts, exudates and resins Proper Labeling on the containers- Name of the Drug, Source & date and category (APPROVED’ or ‘REJECTED)

13 Container should have Batch/lot NoContainer should have Batch/lot No. Name of the Drug Date of procurement Identified By: Procedure First In First Out Separate section for Rejected raw materials Stock Register Packaging Material Separate space for bottles, jars, capsules etc. Containers and closure lids should be proper cleaned and dried before packing the products. Cleaning Testing Storage Controls on printed materials to avoid wrong labeling

14 Equipments e.g. Working Space Nature, number and sizesAdequate for orderly placement of equipments and materials. To facilitate easy and safe working. To minimize/ eliminate risk of mix-ups and cross contamination . Equipments Nature, number and sizes Installation and maintenance record Cleaning SOPs and Records (logbook) Operation SOPs e.g. Anjana/ Pisti: Kharel/ ball mill, sieves/ shifter Churna: Grinder/ disintegrator/ pulverizer, powder mixer, sieves/ shifter Ark: Maceration tank, distillation plant, liquid filling tank with filter/ filter press, visual inspection box

15 Batch Manufacturing Records Each batch manufacturing record of every medicine List of raw material used, quantity obtained from the store Test conducted during the various stages of manufacture. Finished Goods Store Stock area of Storage containing Proper shelves & racks Proper packing and Labeling of finished product. Approved Finished Goods by Quality Control Labs. Specific storage conditions

16 Quality Control Laboratory

17 GLP Personnel: To be headed by an independent person. Duties:GLP is concerned with the organizational process and conditions with which the Laboratory studies are planed, performed, monitored, recorded and reported. Personnel: To be headed by an independent person. Duties: To prepare specifications and testing methods for raw materials and finished products. To sample, test, approve or reject RMs, PMs, semi-finished product and finished products. To supervise and monitor the adequacy of storage conditions. Maintenance of the records of each process where testing of finished product is not possible. Records: Batch Manufacturing Records (BMR) Distribution Records (to facilitate recall) Record of Market Complaints and Adverse Drug Reactions Shelf-life

18 Quality Control, Legal Aspects & Documentation1. Quality Perspectives of Ayurvedic-herbal Products: Status of quality standards for Ayurvedic-herbal products. Databases for identification and authentication of materials. Chemo-profiling & bio-profiling of Ayurvedic-herbal products. Protocols for establishing purity of materials, identification of adulterants, substitutes, pathogenic bacteria & fungi, heavy metals and pesticide residues. Validation of traditional claims through scientific studies and clinical trials. 2. Quality Control: Control of manufacturing processes, statistical quality control, control charts, sampling plans, automated process controls, dosage form controls, Testing programs methods. Product identification systems,  adulteration & misbranding. Maintenance of records. Bioavailability & bio-equivalence. Manufacturer’s reliability. Manufacturer/drug information profile.      

19 processing, packaging and holding of drugsprocessing, packaging and holding of drugs. Control of Components, containers and closures. Packaging & labeling controls. Inspection for compliance with GMP. Potable water standards. Premises- design, construction, maintenance, equipments. Warehousing. 4. Pharmaceutical Process Validation:  Regulatory basis, validation of sterile products & non-sterile products and processes thereof. Analytical method validation. Validation of computer assisted processes. 5. Drug Regulatory Aspects: National and international bodies for drug regulation. Federal food, drug and cosmetic acts along with recent amendments. New drug application, drug efficacy study, implementation review, OTC drug review, drug listing. Drug recalls, product liability, clinical trials. ICH guidelines. WHO & ISO certification. Patents, copyright & trade marks. 6. Documentation: Relevance and importance of documentation, statuary requirements and procedure for documentation, critical examination of documents.    

20 ANALYTICAL SPECIFICATIONS OF SINGLE PLANT MATERIAL/ FormulationsTests Description Colour Odour, Taste Loss on drying at 105oC/ Moisture Content Total- ash Acid insoluble ash Water Soluble Extractive Alcohol Soluble Extractive Total solid pH Value Volatile oil Particle size Bulk density, Tap density TLC/HPTLC/GLC/HPLC/LC-MS Uniformity of weight Disintegration time/Dissolution Time/Friability (if tablet)/Hardness (if tablet) Biological marker Preservative, Binders, Diluents Chelating agent Optimum effective dose Test for heavy/toxic metals Lead, Cadmium, Mercury, Test for Arsenic Pesticide residue Organo chlorine pesticides, organophosphorus pesticides, Pyrethroids Microbial contamination: Total viable aerobic count, Enterobacteriaceae Total fungal count Test for specific Pathogen E coli, Salmonella spp., S.aureus ,Pseudomonas aeruginosa Aflatoxins ( B1,B2,G1,G2 ) Shelf life Tests Passport data of plant material Macroscopic Microscopic Powder analysis Loss on drying at 105 0C/Moisture content pH Value (10% aqueous extract) Total -ash Acid - insoluble ash Water Soluble Extractive Alcohol Soluble Extractive Volatile oils(if oil bearing plants) TLC/ HPTLC/GLC/HPLC/LC-MS Assay for active constituents (total tannins/total alkaloids etc.) Test for heavy/toxic metals (see Annexure-1) Lead, Cadmium, Mercury, Test for Arsenic Pesticide residue (see Annexure-2) Organo chlorine pesticides organophosphorus pesticides, Pyrethroids Microbial contamination (see Annexure-3) Total viable aerobic count Enterobacteriaceae Total fungal count Test for specific Pathogen (see Annexure-3) E coli, Salmonella spp., S.aureus Pseudomonas aeruginosa Aflatoxins ( B1,B2,G1,G2 ) Shelf life

21 Test for heavy/toxic metals As per WHO / FDA (Permissible limit) Lead 10.0 ppm Cadmium 0.30 ppm Mercury 1.00 ppm Arsenic Microbial Load Permissible Limit As per WHO For contamination in the crude plant materials For plant materials that have been pretreated ( used as topical doses form ) For other plant materials for internal use Total Viable Aerobic Count - <107cfug-1 <105cfug-1 E.coli 104g-1 102g-1 10g-1 Total Yeast & mould count 105g-1 103g-1 Total enterobacteriaceae Salmonellae spp. None none S.aureus absent Pseudomonas aeruginosa Coliforms Name of Pesticides/insecticides Limit as per FDA/ EP Quinolphos 0.01 ppm DDE 1.00 ppm Alderin 0.05 ppm Dieldrin DDT HCH (Hexa chlorocyclohexane) 0.30 ppm Malathion 0.10 ppm Parathion Permissible Limits of Aflatoxins B1 0.5 ppm B2 0.1ppm G1 0.5ppm G2

22 PHARMACOGNOSY SECTIONS. No. Name of equipment 1. Microscope Binocular 2. Dissecting Microscope 3. Microtome 4. Physical Balance 5. Aluminum Slide trays 6. Stage Micrometer 7. Camera Lucida(Prism and Mirror type) 8. Chemicals, Glassware’s etc.

23 CHEMISTRY SECTION S. No. Name of equipment 1.Alcohol Determination Apparatus 8. Tablet disintegration apparatus 2. Volatile Oil Determination Apparatus 9. Moisture & temperature Meter/I.R. moisture balance 3. Boiling Point Determination Apparatus 10. Muffle furnace 4. Melting point determination apparatus 11. Electronic balance 5. Digital Refract meter 12. Magnetic stirrer 6. Digital Polari meter 13. Hot air oven 7. Viscometer 14. Refrigerator

24 S. No. Name of equipment 15. Glass/Steel distillation apparatus 25. Limit test apparatus 16. LPG gas cylinders with burners 26. Vacuum rotator evaporator 17. Water bath(Temperature controlled) 27. Deep freezer 18. Heating mantles/hot plate 28. Digital Colorimeter 19. HPTLC apparatus with all accessories 29. HPLC with PDA, ELSD, Floresense/RI detectors 20. Paper chromatography apparatus with accessories 30. GC with FID, ECD 21. Sieve size 10to 120 with sieve shaker 31. LCMS/MS 22. Centrifuge machine 32. AAS 23. Dehumidifier 33. HPTLC with accessories 24. Digital pH meter 34. Rotary vacuum evaporator

25 MICROBIOLOGY S. No. Name of equipment 1. Autoclave sterilizer 2.Laminar air flow apparatus 3. Exhaust fan 4. Sterility testing equipments 5. BOD incubator 6. Automatic micro pipettes 7. Colony counter

26 MINI PHARMACY S. No. Name of equipment 1. Ball mill sieves 11.Thermometer 2. Grinder 12. Capsule filling machine 3. Mini pulverize 13. Electronic balance 4. Disintegrator 14. Tube filling machine (for ointment)and sealing machine 5. Powder & Mass mixer 15. Crimping machine/ointment mixer 6. Granulator drier 16. Liquid filling machine 7. Tablet compressing machine 17. Maceration tank 8. Pill /Vatti cutting machine 18. Dissolution apparatus 9. Sugar coating or polishing pan 19. Tablet hardness tester 10. Hygrometer 20. Friability test apparatus

27 21. Water deionizer plant 27. Stainless steel mixing/storage tank 22. Multi mill 28. Cap sealing machine 23. Tablet coating pan 29. Plate filter press machine 24. Tray drier 30. Humidity/Stability chamber 25. Automatic strip packing machine 31. Bulk density apparatus 26. High speed dissolver and stirrer machine

28 Method Validation Process of documenting or proving that :Developed Analytical Method provides analytical data for the intended use Basic concept of validation process encompasses : The problem and the data requirements The method and its performance characteristics

29 General Process Of ValidationLinearity Accuracy Range Precision / Reproducibility Limit of detection Specificity / Selectivity CANDIDATE METHOD LOQ Robustness / Ruggedness Validated Method Quantitative measurements Collaborative tests Quality Control Procedures Method of known accuracy Validated / Evaluated Method

30 Development & Validation of new HPLC MethodsEquipment: The HPLC (Waters model 600E system, Waters, Milford, MA, USA) equipped with Purospher®-Star RP-18e column (4.6mm i.d.×250 mm, 5 µm, Merck, Darmstadt, Germany), a photo-diode array detector (PDA) (Waters 2996), an inline-degasser AF (Waters), a 20 µL loop manual injector and Waters empower software, was used for analysis of hydroxyanthraquinone derivatives. Mobile Phase: Acetonitrile:methanol (95:5, v/v) (solvent A) and water:acetic acid (99.9:0.1, v/v, pH3.5) (solvent B) were used as mobile phase with a linear gradient elution as follows: 0–15 min, 20% A; 15–25 min, 50% A; 25–30 min, 70% A; 30–40 min, 100% A; at a Flow rate: 0.8 mL/min. PDA Detector: The detection wavelength was set at 290 nm. Column temperature : 30°C Injection volume: 20 µL

31 Repeatability: intra-day and interdayCompound Linearity range (µg/mL) Linear equation r2 Detection limit (ng/mL) Quantitation limit (µg/mL) Emodin-glycoside 20-100 y = x 0.9993 0.78 0.11 Chrysophanol-glycoside 60-200 y = 5101x-69864 0.9979 0.89 0.16 Emodin 1-100 y = 65718x 0.9991 0.56 0.24 Chrysophanol 10-100 y = 35034x 0.9901 0.68 0.26 Physcion 25-400 y = 16100x 0.9959 3.50 0.35 Repeatability: intra-day and interday RSDs of chromatographic determination were observed in the range of 0.23–4.98% and 0.98–4.65%, respectively. results showed good precision of the method Recovery: 95.7–103.5% Robustness / Ruggedness Colum temp. Variation : less than 1% Mobile phase single component variation (AA 0.1 to 1%) : less than 1%

32 Quantification of Hydroxy anthaquinones in the rhizomes of Rheum emodi by HPLC AU 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 Minutes 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 1 2 3 4 5 AU 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 Minutes 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 1 3 4 2 5 Location Emodin glycoside 1 (%) Chrysophanol glycoside 2 (%) Emodin 3 (%) Chrysophanol 4 Physcion 5 L1 0.51 2.23 0.90 1.44 0.19 L2 0.44 1.56 0.49 0.99 0.15 L3 0.58 1.98 0.64 0.88 0.16 Subash C Verma, et.al “Journal of Chromatography A, 1097 (2005)

33 Subash C Verma, et.al Journal of Separation Science, 30 (2007) 15-202. Quantification of compounds Phenolic Compounds (1–10) in ethanolic extract of V. planifolia HPLC chromatographic separation of a mixture of standard phenolic compounds (1–21). Peaks: 1 = 4-hydroxy benzylalcohol; 2 = vanillyl alcohol; 3 = 3,4-dihydroxybenzaldehyde; 4 = 4-hydroxybenzoic acid; 5 = vanillic acid; 6 = 4-hydroxybenzaldehyde; 7 = vanillin; 8 = p-coumaric acid; 9 = ferulic acid; 10 = piperonal; 11 = isovanillin; 12 = syringaldehyde; 13 = acetovanillone; 14 = m-coumaric acid; 15 = veratraldehyde; 16 = ethyl vanillin; 17 = 2,4-dihydroxy acetophenone; 18 = o-vanillin; 19 = coumarin; 20 = p-anisaldehyde; and 21 = eugenol Vanilline : 1.035%, vanillyl Alcohal: 0.526% Vanillic Acid: 0.10% Subash C Verma, et.al Journal of Separation Science, 30 (2007) 15-20

34 TLC Fingerprint of Triphala254 nm 366 nm Chebulinic acid, ellagic acid, and gallic acid (increasing Rf) 2. Terminalia bellerica whole fruit 3. Terminalia chebula whole fruit 4. Phyllanthus emblica 5. Triphala powder 1 2 3 4 5 Mobile phase: Ethyl formate: Toluene: Formic Acid: Water (30:1.5:4:3, v/v) Derivatize: NP Reagent (Diphenyl boric acid amino ethyl ester)

35 Qualification, calibration, validation and maintenanceGeneral Rules for Laboratories Operating under Good Laboratory Practice (GLP) Company Name and Department Page 8 of 17 Version No.: Attachments: none Effective date: DD/MM/YYYY Qualification, calibration, validation and maintenance Equipment shall be adequately inspected, cleaned, and maintained. Equipment used for generation, measurement, or assessment of data shall be adequately tested, calibrated and/or standardized. These activities are frequently called qualification for equipment hardware and single modules and validation for software and complete systems. A schedule shall be established for such operations based on manufacturer’s recommendations and laboratory experience. Time interval for calibration, re-validation and testing The frequency for calibration, re-validation and testing (performance verification) depends on the instrument itself, the recommendations from manufacturers of the equipment, laboratory experience, and the extent of use. E.g. a pH meter should be calibrated before each use and the wavelength of an HPLC variable wavelength detector should be calibrated about every month or whenever the cell is removed and reinstalled. Typically proof of chromatographic instrument performance should be done every 6 to 12 months.

36 Equipment records and other documentsWritten records shall be maintained of all inspection, maintenance, testing, calibrating and/or qualification / validation operations. These records, containing the date of operation, shall describe whether the maintenance operations followed written SOPs. Written records shall be kept of non-routine repairs performed on equipment as a result of failure and malfunction. Such records shall document the nature of the defect, how and when the defect was discovered, and any remedial action taken in response to the defect. Written records may be in log books especially designed for that purpose. A log book should accompany the instrument when it is moved. Remedial action should include a review of effects on data generated before the defect was discovered. Such equipment records should be maintained as long as the data generated by the equipment. Equipment records should include for example: • name of the equipment • name of the manufacturer • model or type for identification

37 Safety Guidelines Safety depends on the maintenance of laboratory discipline. Safety instructions, both general and specific, should be imparted to every new member of the staff and should be regularly supplemented. The general guidelines for safe working include: Prevention of smoking, eating and drinking in the laboratory. Knowledge about the use of the fire fighting equipments and location of emergency exits. Use of laboratory coats and other protective clothing including rubber gloves, ear plugs, safety shoes. Full labeling of all containers of chemicals, warning to be included in case of poisonous and flammable chemicals. Observation of safety rules in handling cylinders of compressed gases. Provision of first-aid materials and instructions and use of antidotes. Laboratory services (air, gas, water) should be turned off when not in use No one should work alone in the laboratory. Unauthorized analysis or experiments should not be allowed in the laboratory. When lifting heavy objects, use legs, instead of back.

38 SYSTEMS AND PROCEDURES IN A QUALITY CONTROL LABORATORYRecords of temperature and humidity of critical areas of the laboratory. Laboratory safety guidelines. Opening and closing of the laboratory. Validation (instruments, personnel and analytical procedures). Regular job based training and evaluation of quality control personnel. Possible evidence that the system continue to perform reliably and reproducibly. Analyst data validation. Periodic validation of analytical methods in use. Periodic validation of analyst. No dispatches without approval of quality control department. Continuous upgrading of quality system. Continuous upgrading of quality standards. Good Laboratory practices. General procedures for analysis abstracted from IP/BP/USP. Current approved and authorized specifications and standard test procedures. Authorized documents for analysis. Retention of analyzed samples. Reference substances and certified working standards and their periodic update. Volumetric solutions and reagents and their standardization schedule. Reporting system for each sample. Analytical reference number. Calibration of instruments, maintenance of log book and calibration schedule of each instrument. Handling of product complaints. Cleaning of laboratory glassware.

39 PLANNING WORK IN THE LABORATORYOften there is inordinate delay in reporting of samples; probably number of samples exceeds the analysis time thereby causing analysis delay and cost. Following points may be of help to the analyst. Proper selection of the method taking into consideration the nature and purpose of analysis and facilities available and to decide whether accuracy and precision are really important in given situation. When a particular procedure fails, when should be analysis be terminated to try another method. The samples may be grouped for analysis such as samples containing vitamins for high throughput. Possible use of Quick Assessment System by which approximate idea about the quality of the drug (90-110% of claim) can be assessed. For high throughput, should use Quick Assessment Procedure e.g. semi-quantitative TLC.

40 Quality Assurance Q C is an operation undertaken in a laboratory to analyze a sample or material within the known probability limits of accuracy and precesion while Q.A. is defined as system through which laboratory conducts the analysis , the proof of which lies in documentation . Corrective Actions are taken under the ‘Act’ when any batch of drug fails during inspection.

41 DOCUMENTATION IS DESIGNED TO ACHIEVE FOLLOWING OBJECTIVESAs a proof that quality control operation or analysis is done. The details of analysis include specifications followed, calibration done. If any, or any other information relevant to work. To ensure the accountability of data . It means that records indicate the results pertain to that sample only which customer has supplied. To ensure traceability of the reported data without wasting time on a future date . If so required. To ensure that adequate precautions are taken against the falsification of data i.e. data cannot be easily tampered. It calls for bounded notebook duly numbered and recording of raw data therein, instead of loose sheets. Proper control of record book both after and during the use and its safe custudy. A proof of replacing testing, using the same or different methods to ensure the quality of the results provided to the clients by implementing checks.

42 Q: Is the Herbal Product Safe Today?Use of modern analytical techniques (especially HPTLC, GC, GCMS, HPLC, CE, FT-IR, UV-VIS, LCMS/MS, AAS and ICPMS to characterize and quantify herbal medicine. Quality assurance also requires to control of starting materials, storage and processing. For this region , an appropriate quality assurance system should be applied in the manufacture of herbal medicines. Q: Is the Herbal Product Safe Today?

43 THANK YOU References: www.ipapharma.org/schedule%20T.pdf THANK YOU