Optimisation of LCMS Techniques for Complementary Medicines

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1 Optimisation of LCMS Techniques for Complementary Medicines Ashley Dowell Southern Cross Plant Science Southern Cross University

2 Complementary Medicines Complementary medicines vitas erals herbal medicines aromatherapy homoeopathics The word complementary indicates that these medicines are not a source of primary healthcare but act with conventional healthcare such as pharmacy. Complementary medicines may be either listed or registered drugs depending on their ingredients and the claims made. In Australia CM s are regulated within the same regulatory framework as conventional pharmaceuticals. As such commercial CM products are required to comply with the same quality control processes as pharmaceuticals. CM s are often sold as complex formulations containing a variety of herbs, vita sand erals. This creates significant challenges for the analytical chemists required to apply quality control of these products.

Stability Testing of Complementary Medicines In 29 the Australian TGA adopted the PIC/S code as its guiding GMP document which requires stability evaluation of complementary medicines and on-going

3 Stability Testing of Complementary Medicines In 29 the Australian TGA adopted the PIC/S code as its guiding GMP document which requires stability evaluation of complementary medicines and on-going shelf life testing for the duration of the product marketing. The result of this is a sharp rise in sponsors seeking stability testing of both new and existing products in order to substantiate the expiry dates on product labels.

4 Analytical Techniques for Stability Evaluation of Complementary Medicines Establish what to measure and monitor Plant extracts are chemically complex requiring analytical techniques which can be stability indicating. UV-Spectrophotometry - useful for single component products but generally non-specific for herbal complexes. Thin Layer Chromatography (TLC) good for quick herbal ID, but lacks detailed chemical information HPLC - PDA is very useful for profiling of herbal products, but becomes complicated with multiple herbal ingredients. LCMS - able to de-convolute complex herbal mixtures. Allows identification and quantitation multi-herbal products.

5 Stability Applications for LCMS LCMS can be applied for both compound identification and quantitation

6 What is LCMS? LCMS - Liquid chromatography Mass spectrometry. Analytical chemistry technique HPLC combined with a Mass Selective Detector LC-MS is a powerful technique used for many applications which has very high sensitivity and selectivity. Generally applied towards detection and identification of chemicals in the presence of other chemicals (in a complex mixture). Mass spectrometry (MS) is an analytical technique that measures the massto-charge ratio of charged particles. MS works by ionizing chemical compounds to generate charged molecules or molecule fragments and measuring their mass-to-charge ratios. In our laboratory we use a single-quadrupole mass spectrometer typically fitted with an APCI (Atmospheric Pressure Chemical Ionisation)detector. For natural products APCI is a comprehensive interface however for larger molecules such as tannins ESI (Electron Spray Ionisation) interface is more sensitive.

7 Herbal Compound Identification using LCMS. Marker compound selection Confirmation of specificity Assignation of marker compound co-ordinate (m/z : retention time)

8 Olive Leaf Traditional Greek medicine used for immune stimulation The product contains many active principles, but the major active constituent is a secoiridoid called Oleuropein. 8 7 MSD1 TIC, MS File (64DATA\6427\CPA689.D) APCI, Pos, Scan, Frag: 15, "pos 15" 1 *MSD1 SPC, time=9.622:9.784 of 64DATA\6427\CPA689.D APCI, Pos, Scan, Max: Olive leaf MS-TIC Oleuropein MSD1 558, EIC=557.7:558.7 (64DATA\6427\CPA689.D) APCI, Pos, Scan, Frag: 15, "pos 15" 35 3 Olive leaf EI m/z

124. 137.2 152.4 152. 153.2 661.2 445.2 66. 165.2 123.2 331.2 465.2 135.2 133.2 Pau D arco Pau D arco (Bow Wood) - Tabebuia (syn.

$-Actives - napthoquinones derived from Lapachol. MSD1 TIC, MS File (64DATA\6427\CPA69.$ $D APCI, Pos, S 15 1 5 5 6 7 8 9 1 11 12 13 14 MSD1 465, EIC=464.7:465.7 (64DATA\6427\CPA69.$

9 Pau D arco Pau D arco (Bow Wood) - Tabebuia (syn. Hydroanthus) impetiginosa. - Inner bark is used in traditional medicines - expectorant and for treating influenza. -Actives - napthoquinones derived from Lapachol. MSD1 TIC, MS File (64DATA\6427\CPA69.D) APCI, Pos, Scan, Frag: 15, "pos 15" 25 2 Pau d'arco MS-TIC *MSD1 SPC, time=1.47:1.614 of 64DATA\6427\CPA69.D APCI, Pos, S MSD1 465, EIC=464.7:465.7 (64DATA\6427\CPA69.D) APCI, Pos, Scan, Frag: 15, "pos 15" Pau d'arco EI MSD1 66, EIC=659.7:66.7 (64DATA\6427\CPA69.D) APCI, Pos, Scan, Frag: 15, "pos 15" Pau d'arco EI Pau d'arco marker compound Max: m/z

$methoxyflavone derivatives known as astragalosides. 447.2 MSD1 TIC, MS File (64DATA\6427\CPA691.$

10 Astragalus Astragalus membranaceus (huáng qí) - herbaceous plant - roots used in TCM - reinforces qi and invigorates the spleen - contains methoxyflavone derivatives known as astragalosides MSD1 TIC, MS File (64DATA\6427\CPA691.D) APCI, Pos, Scan, Frag: 15, "pos 15" 1 *MSD1 SPC, time=7.329:7.59 of 64DATA\6427\CPA691.D APCI, Pos, Sca Max: Astragalus MS-TIC 8 Astragaloside MS MSD1 447, EIC=446.7:447.7 (64DATA\6427\CPA691.D) APCI, Pos, Scan, Frag: 15, "pos 15" Astragalus EI m/z

11 Marker Ion Co-ordinates Herbal ingredient Selected Marker Ion MW+H RT () Olive leaf (Olea europaea) Astragalus (Astragalus membranaceus) Pau d arco (Tabebuia impetiginosa)

12 Marker Ion Specificity Olive Leaf MSD1 TIC, MS File (64DATA\6427\CPA689.D) APCI, Pos, Scan, Frag: 15, "pos 15" Sorbic acid peak Olive leaf marker ion check MSD1 558, EIC=557.7:558.7 (64DATA\6427\CPA689.D) APCI, Pos, Scan, Frag: 15, "pos 15" MSD1 447, EIC=446.7:447.7 (64DATA\6427\CPA689.D) APCI, Pos, Scan, Frag: 15, "pos 15" MSD1 465, EIC=464.7:465.7 (64DATA\6427\CPA689.D) APCI, Pos, Scan, Frag: 15, "pos 15" EI m/z 588 EI m/z 447 EI m/z

$Specificity Pau D arco MSD1 TIC, MS File (64DATA\6427\CPA69.D) APCI, Pos, Scan, Frag: 15, "pos 15" 25 2 15 Pau d'arco marker ion check 1 5 5 6 7 8 9 1 11 12 13 14 MSD1 558, EIC=557.7:558.$

13 Specificity Pau D arco MSD1 TIC, MS File (64DATA\6427\CPA69.D) APCI, Pos, Scan, Frag: 15, "pos 15" Pau d'arco marker ion check MSD1 558, EIC=557.7:558.7 (64DATA\6427\CPA69.D) APCI, Pos, Scan, Frag: 15, "pos 15" EI m/z MSD1 447, EIC=446.7:447.7 (64DATA\6427\CPA69.D) APCI, Pos, Scan, Frag: 15, "pos 15" EI m/z MSD1 465, EIC=464.7:465.7 (64DATA\6427\CPA69.D) APCI, Pos, Scan, Frag: 15, "pos 15" EI m/z

$Specificity - Astragalus MSD1 TIC, MS File (64DATA\6427\CPA691.D) APCI, Pos, Scan, Frag: 15, "pos 15" 2 175 15 125 1 75 5 25 Astragalus marker ion check 5 6 7 8 9 1 11 12 13 14 MSD1 558, EIC=557.$

14 Specificity - Astragalus MSD1 TIC, MS File (64DATA\6427\CPA691.D) APCI, Pos, Scan, Frag: 15, "pos 15" Astragalus marker ion check MSD1 558, EIC=557.7:558.7 (64DATA\6427\CPA691.D) APCI, Pos, Scan, Frag: 15, "pos 15" EI m/z MSD1 447, EIC=446.7:447.7 (64DATA\6427\CPA691.D) APCI, Pos, Scan, Frag: 15, "pos 15" EI m/z MSD1 465, EIC=464.7:465.7 (64DATA\6427\CPA691.D) APCI, Pos, Scan, Frag: 15, "pos 15" EI m/z

15 Reference Sample MSD1 TIC, MS File (64DATA\6427\RFS45.D) APCI, Pos, Scan, Frag: 15, "pos 15" Reference mixture prepared from raw materials used by manufacturer Rutin peak MSD1 588, EIC=587.7:588.7 (64DATA\6427\RFS45.D) APCI, Pos, Scan, Frag: 15, "pos 15" 588, 9.5 = oleuropein = olive leaf MSD1 447, EIC=446.7:447.7 (64DATA\6427\RFS45.D) APCI, Pos, Scan, Frag: 15, "pos 15" , 7.3 = astragaloside = astragalus membranecus MSD1 465, EIC=464.7:465.7 (64DATA\6427\RFS45.D) APCI, Pos, Scan, Frag: 15, "pos 15" , 1.5 = pau d'arco marker = pau d'arco

16 Comparison to reference Sample MSD1 TIC, MS File (64DATA\6427\CPA637.D) APCI, Pos, Scan, Frag: 15, "pos 15" Indicates: Different species of the same genus 12 ( eg. Arnica montana with 1 Arnica 8 chamissonis) Mixed Herbal Product MSD1 588, EIC=587.7:588.7 (64DATA\6427\CPA637.D) APCI, Pos, Scan, Frag: 15, "pos 15" 558, 9.5 = oleuropein = olive leaf Glycine max sample 4 2 Different extraction technique 16 (7:3 14 ethanol:water vs :7 ethanol: water) MSD1 447, EIC=446.7:447.7 (64DATA\6427\CPA637.D) APCI, Pos, Scan, Frag: 15, "pos 15" 2 Creation of processing artefacts (Bacosides 15 B in Bacopa monnieri, , 7.4 = astragaloside = astragalus membranecus Glycine max reference MSD1 465, EIC=464.7:465.7 (64DATA\6427\CPA637.D) APCI, Pos, Scan, Frag: 15, "pos 15" 465, 1.5 = pau d'arco marker = pau d'arco

$2 163.2 425.2 49.2 *MSD1 SPC, time=1.271:1.524 of 65DATA\65 *MSD1 SPC, time=14.929:15.128 of 65DATA\653\34-41.$ $D APCI, Pos, Sc *MSD1 SPC, time=14.514:14.713 of 65DATA\653\34-41.$

17 Quantitative Analysis - Ginkgolides - by LCMS *MSD1 SPC, time=1.271:1.524 of 65DATA\65 *MSD1 SPC, time=14.929: of 65DATA\653\34-41.D APCI, Pos, Sc *MSD1 SPC, time=14.514: of 65DATA\653\34-41.D APCI, Pos, Scan, Frag: 15, "p 1 Max: Max: Max: Horse chestnut 8 bilobalide 8 ginkgolide B 8 ginkgolide A Aescin 2 4 m/z m/z m/z

18 MSD1 TIC, MS File (65DATA\653\34-41.D) APCI, Pos, Scan, Frag: 15, "pos 15" Ginkgolide Standard Mix compound Marker Ion MW+H RT () MSD1 163, EIC=162.7:163.7 (65DATA\653\34-41.D) APCI, Pos, Scan, Frag: 15, "pos 15" 4 bilobalide bilobalide MSD1 425, EIC=424.7:425.7 (65DATA\653\34-41.D) APCI, Pos, Scan, Frag: 15, "pos 15" ginkgolide J ginkgolide B MSD1 441, EIC=44.7:441.7 (65DATA\653\34-41.D) APCI, Pos, Scan, Frag: 15, "pos 15" ginkgolide C MSD1 49, EIC=48.7:49.7 (65DATA\653\34-41.D) APCI, Pos, Scan, Frag: 15, "pos 15" ginkgolide J ginkgolide C ginkgolide A ginkgolide A ginkgolide B

19 Complications with LCMS for stability indicating analysis MSD1 TIC, MSFile (L:\LCMS-1\DATA21LCMS\16DATA\1615A\1141.D) APCI, Pos, SIM, Frag: 15, "Positive 15" Allantoin Pyrrolizidine alkaloids 12 Lycopsae

Summary LCMS is a useful technique for compound identification in stability studies of Complementary Medicines but careful consideration must be given to the dynamic nature of MS detector conditions.

20 Summary LCMS is a useful technique for compound identification in stability studies of Complementary Medicines but careful consideration must be given to the dynamic nature of MS detector conditions. To qualify this it is important to run reference samples to confirm observations. LCMS is a powerful tool for quantitation of herbal actives in complex formulations but calibration co-efficients must be validated and the linear range well defined. When applying MS quantitation for a class of compounds where only one standard is available the reality of differential response must be considered and results reviewed in this context.

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