HPTLC. For the Identification of Plant Materials. Seoul February Eike Reich CAMAG Laboratory

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1 HPTLC For the Identification of Plant Materials Seoul February 2012 Eike Reich CAMAG Laboratory

2 2 Who we are: CAMAG Laboratory CAMAG Muttenz; CSI Wilmington (NC, USA) Partners: ANCHROM (India); Nikyang (China) Fundamental and applied research Publications, education, training ( Method development and method validation Feasibility studies / contract analyses Support for students; diploma, masters, Ph.D. Collaboration with Pharmacopeias of Switzerland (PhHelv), Europe (PhEur), and United States (USP)

3 3 The Team

4 4 What is HPTLC? IN! High Performance Thin-Layer Chromatography TLC for the 21st century Instrumental TLC Application Development Documentation Densitometry Truly plug and play Fully cgmp compliant A new concept Instruments Scientific basis Standardized methodology Validated methods

5 5 Standardized basic equipment Clear specification of HPTLC plates (type, format, manufacturer) Software control for absolute reproducibility of all parameters Independence from environmental factors (humidity!) Eliminating human factors Emulation by manual operation still possible to a certain degree

6 6 HPTLC and GMP All instruments can be IQ, OQ, PQ qualified Software can be qualified (also 21CFR part 11) SOPs are available Methods must be fit for purpose Also qualitative methods can be validated Training for personnel is available

7 7 TLC / HPTLC: the off-line principle Application The plate Development Detection and quantitative Evaluation Documentation

8 8 Standardization of methodology Plate setup and handling Sample application (as band) Chamber geometry and saturation Humidity control Developing distance Derivatization procedure Documentation (electronic images) Evaluation SOP A Standardized Approach to Modern High-Performance Thin-Layer Chromatography (HPTLC). Reich, E., Schibli, A., (2004) J. Planar Chromatogr. 17,

9 9 Method template

10 Identification of Thyme leaf 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Track Volume Sample Track Volume Sample 1 7 µl Thyme leaf #1 9 10 µl Oregano leaf #1 2 10 µl Thyme leaf #1 10 15 µl Oregano leaf #1

10 10 Identification of Thyme leaf Track Volume Sample Track Volume Sample 1 7 µl Thyme leaf # µl Oregano leaf # µl Thyme leaf # µl Oregano leaf # µl Thyme leaf # µl Oregano leaf # µl Thyme leaf # µl Oregano leaf # µl Wild thyme leaf # µl Oregano leaf # µl Wild thyme leaf # µl Oregano leaf #5 7 2 µl Rutin, Rosmarinic acid 15 Blank 8 7 µl Oregano leaf #1

11 11 Examples from other labs (HS Wädenswil) Oswego tea

12 12 Examples from other labs (Uni Regensburg) Coptis rhiz.

found in EP The USP Dietary Supplement Compendium TLC and state of the art HPTLC Chinese

13 13 Sources of methods European Pharmacopoeia (EP) New monographs feature TLC and HPTLC in parallel British, French, German, Swiss Pharmacopoeias Offer specific monographs not found in EP The USP Dietary Supplement Compendium TLC and state of the art HPTLC Chinese Pharmacopoeia HPTLC atlas as a supplement of 2005 ed. Japanese Pharmacopoeia Only simple TLC

Indian Herbal Pharmacopoeia (54 monographs) Quality Standards of

14 14 More sources of methods American Herbal Pharmacopoeia (19 monographs) Quality Standards of Indian Medicinal Plants (8 volumes) Indian Herbal Pharmacopoeia (54 monographs) Quality Standards of Traditional Chinese Medicines (Chinese only) Wagner, H. and Bladt, S. Plant Drug Analysis

15 15 Reality check for official/literature methods: Methods are often Outdated (silica gel G!) Tedious (e.g. 2h soxhlett, evaporation, dissolution) Not fit for purpose (primary geared towards assay) Not standardized / harmonized (who has seen the described results and when? Not validated (but treated as if they would be) Vague in result description (additional zones )

16 16 Converting existing TLC methods to HPTLC Assumptions: Results on HPTLC and TLC plates are similar if No changes are made to chromatographic system Same equipment is used Original TLC method was optimized Due to the higher separation power HPTLC plates Usually give improved result Require shorter developing distance less time HPTLC with no instruments looks bad, but so does TLC

17 17 Converting existing TLC methods to HPTLC Practical aspects (I) Do not change chromatographic system (chamber configuration, mobile phase, stationary phase) Reduce application volume (generally) to 1/5 (typically 2 μl) Employ standardized methodology (based on SOP): Fixed (x, y) application positions, (e.g.) 8 mm bands Use 60 mm developing distance Fixed drying time and temperature Use dipping instead of spraying if possible Fixed waiting times between derivatization and evaluation Obtain multiple images (if possible) of plate (e.g. UV 254, UV 366 prior to derivatization, and white light, UV 366 after derivatization

18 18 Converting existing TLC methods to HPTLC Practical aspects (II) Use same samples on TLC and HPTLC Evaluate whether changes in the result can Fall under additional weak zones may be seen Are due to natural variability of plant Color description is always subjective: e.g. blue, bluish blue white blue green, etc. Rf is predictable only for validated methods!

19 19 Example: USP method for chamomile TLC HPTLC application volume 2 μl

20 20 Method optimization: Tribulus terrestris Review of literature Improvement of sample preparation Optimization of detection Comparison of multiple samples

21 21 Tribulus terrestris: collaborating Lab 1 Sample preparation: PhEur Senega root, CP Tribulus terrestris / without chloroform, J. Pharmaceutical Analysis Chromatography: Senega root (3h 5 min)

22 22 Using standards 1 escin 2 protodioscin 3 prototribescin

23 23 Chinese Pharmacopoeia Method

24 24 CP: chloroform dichloromethane 1 escin 2 protodioscin, prototribescin

25 25 Method evaluation CAMAG Laboratory Method Senega root, 1h developing time

26 26 Chinese Pharmacopoeia Method (dichloromethane)

27 27 Other mobile phases / detections: Propanol : ethyl acetate : water : acetic acid (80:60:60:3) Standards: escin, arbutin hydroquinone SulfuricNP/PEG acid anisaldehyde vanillin DMBAreagent

28 28 Other mobile phases / detections: Dichloromethane : methanol : water (70:30:4) Standards: escin, arbutin hydroquinone NP/PEG DMBA Liebermann-Burchart-Reagent

29 29 Other mobile phases / detections: Toluene : ethyl acetate : acetic acid (25:15:10) Standards: escin, arbutin hydroquinone NP/PEG Sulfuric acid anisaldehyde DMBA Liebermann-Burchart-Reagent vanillin-reagent

30 30 Improvement of sample preparation Standard: 0.5 g of powdered sample are mixed with 5 ml of methanol and sonicated for 10 minutes. This solution is then centrifuged for 5 minutes. The supernatant is used as test solution. Modified A: use dichloromethane as extraction solvent Modified B: use acetone as extraction solvent Modified C: use n-butanol as extraction solvent Modified D: 1.5 g of powdered sample are mixed with 5 ml of methanol and sonicated for 10 minutes. This solution is then centrifuged for 5 minutes and the supernatant is transferred into a clean centrifuge tube. 5 ml of n-hexane are added to the solution. The mixture is shaken vigorously and the centrifuged for 5 min. The hexane layer is discarded. Repeat this step, use the methanolic phase as a test soln.

31 31 Improvement of sample preparation EP CP S A B C D

32 32 Final method

33 Additional samples 1 2 3 4 5 6 7 8 9 10 11 12 1:

33 33 Additional samples : escin; 2: arbutin; 3: fruit France; 4: fruit China; 5: fruit India; 6,7: fruit 8: leaf Senegal; 9, leaf France; 10: stem Europe; 11:stem France, 12: stem Senegal

34 34 Developing an ID method from scratch Review literature for related plants Obtain multiple samples form different accessions Obtain samples of related plants and known adulterants Optimize sample preparation and detection Avoid toxic solvents Start with silica gel, select mobile phase Reich, E., Schibli, A.:HPTLC for the analysis of medicinal plants, chapter 5, Thieme 2007

DCM; 5: chloroform; 6: acetone; 7: ethanol; 8: methanol; 9: ethanol-water(7:3); 10:

35 35 Optimization of extraction Non polar mobile phase: toluene, ethyl acetate (95:5) Extraction solvent: 1: heptane; 2: toluene; 3: MTBE; 4: DCM; 5: chloroform; 6: acetone; 7: ethanol; 8: methanol; 9: ethanol-water(7:3); 10: methanol-water (8:2); 11: methanol-acetic acid (9:1); 12: methanol-ammonia 25% (8:2).

36 Optimization of extraction Polar mobile phase: chloroform, methanol,water (70:30:4) 1 2 3 4 5 6 7 8 9 10 11 12 Extraction solvent: 1: heptane; 2: toluene; 3: MTBE; 4:

36 36 Optimization of extraction Polar mobile phase: chloroform, methanol,water (70:30:4) Extraction solvent: 1: heptane; 2: toluene; 3: MTBE; 4: DCM; 5: chloroform; 6: acetone; 7: ethanol; 8: methanol; 9: ethanol-water(7:3); 10: methanol-water (8:2); 11: methanol-acetic acid (9:1); 12: methanol-ammonia 25% (8:2).

37 Optimization of extraction Specific mobile phase: acetonitrile, water, formic acid (30:8:2) 1 2 3 4 5 6 7 8 9 10 11 12 Extraction solvent: 1: heptane; 2: toluene; 3:

37 37 Optimization of extraction Specific mobile phase: acetonitrile, water, formic acid (30:8:2) Extraction solvent: 1: heptane; 2: toluene; 3: MTBE; 4: DCM; 5: chloroform; 6: acetone; 7: ethanol; 8: methanol; 9: ethanol-water(7:3); 10: methanol-water (8:2); 11: methanol-acetic acid (9:1); 12: methanol-ammonia 25% (8:2).

38 Optimization of extraction Specific mobile phase: ethyl acetate, acetic acid, formic acid, water (100:11:11:27) 1 2 3 4 5 6 7 8 9 10 11 12 Extraction solvent: 1: heptane; 2:

38 38 Optimization of extraction Specific mobile phase: ethyl acetate, acetic acid, formic acid, water (100:11:11:27) Extraction solvent: 1: heptane; 2: toluene; 3: MTBE; 4: DCM; 5: chloroform; 6: acetone; 7: ethanol; 8: methanol; 9: ethanol-water(7:3); 10: methanol-water (8:2); 11: methanol-acetic acid (9:1); 12: methanol-ammonia 25% (8:2).

39 Extraction: Stinging Nettle 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1, 2: standards; 3-14

8: isopropanol, 9: ethanol, 10: methanol, 11: methanol-water (8:2), 12: ethanol-water

39 39 Extraction: Stinging Nettle , 2: standards; 3-14 extracts of Nettle obtained with: 3:hexane, 4: toluene, 5: MTBE, 6: DCM, 7: ethyl acetate, 8: isopropanol, 9: ethanol, 10: methanol, 11: methanol-water (8:2), 12: ethanol-water (7:3), 13: ethanol-water (1:1), 14: water; 15 extract of Nettle obtained by reflux with methanol)

40 40 Practical approaches to mobile phases Standard systems Spot test 4-solvent-approach Prisma-model CAMAG - Optimization scheme

water, MEK (50/10/10/30) ethyl acetate, formic acid, water

41 41 Standard systems: example ethyl acetate, formic acid, acetic acid, water (15/1/1/2) ethyl acetate, formic acid, water, MEK (50/10/10/30) ethyl acetate, formic acid, water (80/10/10) ethyl acetate, formic acid, acetic acid, water (100/11/11/26)

42 42 The CAMAG - optimization scheme

43 43 CAMAG - scheme: Example level 1

44 44 CAMAG - scheme: Example level 2

45 45 CAMAG - scheme: Example level 2

46 46 CAMAG - scheme: Example level 3

47 47 CAMAG - scheme: Example level 4

48 48 Comparison of bonded phases Mode switching Normal phase: THF, toluene, formic acid, water (24:12:3:1.5) Silica gel Diol Cyano RP18 W Reversed phase: methanol, formic acid, water (5.5:1:4.5) Cyano RP18 W Vitexin, orientin, isovitexin, isoorientin, chrysin, and 5 Passion Flower samples

49 49 Validation of qualitative methods Validated method Robustness Precision Specificity Validation protocol Stability Optimization Method selection Validation of high-performance thin-layer chromatographic methods for the identification of botanicals in a cgmp environment. Reich, E., Schibli, A., DeBatt, A. (2008) J. AOAC Int. 91,

50 50 Detection of adulteration Wrong plant part Wrong species Addition of API Economic adulteration

51 51 Advantages of planar chromatography: Nearly unlimited choice of stationary and mobile phase combinations Multiple samples can be analyzed in parallel Single use of stationary phase: no clean-up Static two dimensional result: an array of separated sample components Multiple detection without the need to repeat chromatography Chemical / biological / MS detection or extraction Visual evaluation

52 52 Analysis of dietary supplements Golden seal Root Herb Fruit Product 1 Product 2 Product 3 Echinacea purpurea herb Echinacea purpurea root 400 mg Echinacea purpurea flowers 360 mg Hydrastis canadensis root125 mg

53 53 ID and adulteration of Equisetum arvense Track Volume Sample Track Volume Sample 1 3 µl Common horsetail herb 1 9 3µL + 1.5µL Common horsetail herb 3 with 5% Equisetum palustre CRS 2 3 µl Equisetum palustre CRS 10 3µL + 3µL Common horsetail herb 3 with 10% Equisetum palustre CRS 3 3µL + Common horsetail herb 1 with 5% 11 3 µl Rutin 1.5µL Equisetum palustre CRS 4 3µL + Common horsetail herb 1 with 10% 12 3 µl Hyperoside 3µL Equisetum palustre CRS 5 3 µl Common horsetail herb µl Caffeic acid 6 3µL + Common horsetail herb 2 with 5% 14 Blank 1.5µL Equisetum palustre CRS 7 3µL + Common horsetail herb 2 with 10% 15 Blank 3µL Equisetum palustre CRS 8 3 µl Common horsetail herb 3

54 54 Detection of sibutramin by HPTLC UV spectrum UV 254nm UV 225nm Toluene, methanol = 95 : 5

55 Amarant - a red azo dye - as adulterant of Bilberry extract Image evaluated under white light enhanced 1 2 3 1 2 3 1-Butanol,

55 55 Amarant - a red azo dye - as adulterant of Bilberry extract Image evaluated under white light enhanced Butanol, water, formic acid = 40 : 15 : 10 1: Bilberry dry extract 2: Bilberry dry extract spiked with amaranth (spiking level 0.25 %) 3. Amaranth

56 56 Nimisulide in Curcuma 1% 2,5-Dichloro-1,4-benzoquinone reagent

57 57 Ylang Ylang Essential oil: toluene, ethyl acetate = 95 : 5, Anisaldehyde reagent Linolenic acid Sunflower oil Argan oil Various fatty oils: DCM, acetic acid, acetone = 20 : 40 : 50 Phosphomolybdic acid reagent

58 58 Further questions?

STABILITY INDICATING ASSAY. differentiate an intact drug from its potential decomposition products 425.

STABILITY INDICATING ASSAY. differentiate an intact drug from its potential decomposition products 425. .1. INTRODUCTION.1.1 STABILITY INDICATING ASSAY The stability - indicating assay is a method that is employed for the analysis of stability samples in pharmaceutical industry. It is essential to validate