Day 1 - US Pharmacopeial Convention

Impurities, Adulteration, and the Changing 

Role of the USP in Global Drug Quality 

WELCOME 

USP Sponsored Workshop at 2010 AAPS-PSWC Meeting 

November 13-14, 2010 

Morial Convention Center, New Orleans, LA 

Quality Standards for Medicines, Supplements, and Food Ingredients throughout the World 

Workshop Goals and Objectives 

� An update from USP on some key initiatives 

�� Presentations with time for dialog on issues of 

importance to industry, regulators and the USP 

� A chance to network and dialog with colleagues 

dealing with similar issues from different perspectives 

– a learning experience for all of us 

� USP is looking for input from interested parties 

– How should USP be involved? 

– What standards are missing or need updating? 

– What are the roles of industry, regulators and USP – how 

can we work in complementary ways and avoid rework? 

11/23/2010 

1

USP Sponsored Workshop at 2010 AAPS-PSWC Meeting 

Impurities, Adulteration, and the Changing Role of the USP in Global Drug Quality 

November 13-14, 2010 

MMorial i l CConvention ti CCenter, t NNew OOrleans, l LA 

USP’s Elemental Impurities Initiative – Perspectives and 

Current Status 

Anthony J. DeStefano, Ph.D. 

Vice President, General Chapters, USP 


Background-Issues with Chapter Heavy Metals 

� Difficulties in reproducibility 

– Monitor solutions, standards, recovery y issues 

� Difficulties with reagents – safety issues 

– All procedures generate H2S; thioacetamide not allowed in 

California and several European countries 

� Nondiscriminatory screening test 

– Not element specific 

– Sensitivity varies by element 

– Only a few elements respond at required sensitivities 

� Visual comparison test 

– Limits based on visual acuity, not toxicology 

11/23/2010 

2

A Fifteen Year Saga Begins 

� 1995 - Pharmacopieal Forum stimuli article 

identified issues with method II (K. (K 

Blake) 

� 2000- Second stimuli article in 2000 

proposed ICP-MS as an instrumental 

alternative (T. Wang) 

�� 2004 - Lewen Lewen, et al al, JJ. Pharm and Biomed 

Analysis 

Comparisons Between Instrumental Methods and 

“Although still widely accepted and used in 

the pharmaceutical industry industry, these methods 

based on the intensity of the color of sulfide 

precipitation are non-specific, insensitive, 

time-consuming, labor intensive, and more 

often than hoped, yield low recoveries or no 

recoveries at all. all ” 

(Wang, T. et al, J. Pharm. & Biomed. Anal., Vol. 23 

(2000) 867-890) 

11/23/2010 

3

Comparisons Between Instrumental Methods and (Lewen, 

N. et al J. Pharm. & Biomed. Anal. 35 (2004) 739-752) 

Average % Recoveries 

120 

100 

80 

60 

40 

20 

0 

Pb As Se Sn Sb Cd Pd Pt Ag Bi Mo Ru In Hg 

Elements 

The Saga Continues 

USP Results 

ICP-MS Results 

� Post 2005, General Chapters Expert 

Committee appointed a Heavy Metals 

subcommittee 

� Subsequently formed Advisory Panel 

– In depth expertise in methodology and 

toxicology 

– Includes FDA liaisons in both areas 

� Independent Stakeholder Project Team 

initiated 

11/23/2010 

4

Recent Activities 

� USP published a Stimuli Article in 

Pharmacopeial Forum (PF) 34(5) for public 

comment 

� Discussed at Prescription-Nonprescription 

Stakeholder Forum meetings held at USP 

headquarters 

� Institute of Medicine (IOM) meeting in 

August, g 2008 to provide p a non-biased view 

of the methodology and toxicology aspects 

� Workshop with international participation to 

gather further input from stakeholders at 

USP headquarters in April, 2009 

Recent Activities 

� Comments and observations from all activities 

collected and provided to Advisory Panel for their 

deliberations 

� Based on Advisory Panel recommendations, USP 

published draft documents for comment in PF 36(1) 

and put on USP web site: 

– Elements and limits chapter 

– Methodology chapter 

– Dietary Supplement chapter 

– Sti Stimuli li article ti l with ith rationales ti l for f limits li it 

– Stimuli article with responses to comments on PF 34(5) 

stimuli article 

� Follow-up: On USP website - draft chapters with 

current Expert Panel recommendations (to be 

updated - not necessarily what will end up in PF) 

11/23/2010 

5

Some Issues 

� Elements to be controlled 

� Toxicologically-relevant limits 

– Impact of daily dosage 

– Limits expressed as concentrations? 

� Methodology 

– Instrumental 

– Wet chemistry 

� Elements and limits relevant to dietary 

supplements 

� Harmonization with other pharmacopeias 

Design and Scope of Chapters 

� IOM meeting and Advisory Panel meetings 

- important differences between 

pharmaceuticals, dietary supplements and 

foods 

– “Dose” 

– Botanicals 

– Controlling g regulatory g y bodies 

– Patient population 

� Foods represent a special case with many 

additional issues 

11/23/2010 

6

Design and Scope of Chapters 

� Limit scope of required (below ) chapter 

to pharmaceuticals (draft chapter ) 

� Chapter with metals and limits relevant to 

dietary supplements drafted by Dietary 

Supplement General Chapter Expert Committee 

(draft chapter ) 

� Separate chapter for methodology (draft chapter 

) referenced by both and 

Elements – Key Input 

� Outcome from IOM Meeting 

– Critical contaminants are the Lead, Arsenic, 

Mercury and Cadmium – the “Big Four” 

– Consider EMEA catalysts 

� EMEA Guideline on the Specification Limits 

for Residues of Metal Catalysts 

(CPMP/SWP/4446/00) 

– 14 catalysts used in pharmaceutical synthesis 

– Does not address potential contaminants 

– EMEA working party considering addition of 

contaminants to the Guidance 

11/23/2010 

7

Elements – USP Draft Chapter 

� Elements in the environment 

– As, Pb, Cd, Hg 

�� Elements in EMEA Catalyst Guidance 

– Exclude zinc and iron, which are not toxic at levels 

relevant in pharmaceuticals 

� Need to control in drug products if presence is 

possible 

– Deliberately added (catalyst) 

– Possible supply-chain contaminant or adulterant 

– Process issue (equipment) 

� Excipients and APIs – Levels sufficiently low that 

drug product meets permissible daily dose 

requirements, except for Large Volume Parenterals 

Impurities and Limits – Current Panel 

Recommendations 

Oral Daily Parenteral Large Large-Volume Volume 

Element l 

Dose PDE 

(µg/day) 

Daily Dose 

Dose 

PDE (µg/day) 

Parenteral 

Component 

Limit (µg/g) 

Arsenic 15 (EPA) 1.5 0.15 

Cadmium 5 (ATSDR) 0.5 0.05 

Lead 10 (FDA) 1 0.1 

Mercury Mercuryy 15 (EPA) ( ) 

1.5 0.15 

11/23/2010 

8

Impurities and Limits – Current Panel 


Element 

Oral Daily Dose PDE 

(µg/day) 

Parenteral Daily 

Dose PDE 

(µg/day) 

Large Large-Volume Volume- 

Parenteral Component 

Limit (µg/g) 

Chromium 250 25 2.5 

Copper 2500 250 25 

Manganese 2500 250 25 

Molybdenum 250 25 2.5 

Nickel 250 25 2.5 

Palladium 100 10 1.0 

Platinum 100 10 1.0 

Vanadium 

Osmium 

250 25 2.5 

Rhodium 

Ruthenium 

Iridium 

100 

(Combination not to 

exceed) 

10 

(Combination not 

to exceed) 

Limits – Toxicologically Based 

1.0 

(Combination not to 

exceed) 

� EMEA Guidance contains rationale for limits 

�� Extensive databases exist regarding 

toxicology of Hg, Pb, Cd and As 

– IOM participants and Advisory Panel members 

agreed further studies not needed 

� Speciation is an issue, especially for As 

– Highly g y toxic in inorganic g form 

– Nontoxic in some organic forms 

– Should be monograph specific 

11/23/2010 

9

Meeting The Limits – Three Approaches 

� Daily Dose Option 

– Dosage form is analyzed and meets the PDE 

requirements on a daily dose basis 

� Individual Component Option - LVPs 

– Each component of the dose form meets the 

limits established in the LVP column 

� Summation Option 

– Sum the individual component levels per 

equation i in i the h chapter h and d compare to Daily D il 

Dose PDE 

Some Possible Scenarios 

Impurity possible via the nature or source of 

the product (e.g., natural sources, supply 

chain)? )? 

� No - Justify documentation and testing 

requirements with regulatory authorities 

� Yes – Demonstrate control via testing or 

validated process on schedule agreed with 

regulatory authorities 

11/23/2010 

10

Some Possible Scenarios 

Added to the process or a possible inadvertent 

process impurity? 

� No – Testing may ma not be req required ired if absence is 

acceptably demonstrated and documented 

� Yes - Removed via a validated process? 

– If not consistently removed, control as needed 

– If consistently removed, justify documentation and 

testing requirements (if any) with regulatory 

authorities 

� Drug product must pass if tested during product 

life 

� Higher levels – allowed via monograph 

Dietary Supplements – Draft USP Chapter 

�� Draft 2232 limits are identical to draft 

limits 

� Chapter includes a limit for methyl 

mercury 

� Chapter includes methodology for 

speciation of mercury and arsenic 

11/23/2010 

11

Methodology – Draft USP Chapter 

� One or two methods - 4400 monographs 

� Like Residual Solvents, provides 

procedures d as a starting t ti point i t 

� For this chapter, verification = validation 

� Chapter provides minimum validation 

acceptance criteria 

– Embodiment of for validation and 

demonstration of equivalence q to USP procedures 

p 

� Dietary supplements chapter refers to this 

chapter for all but methyl mercury and 

speciation issues 

Advisory Panel Discussed Potential Detection Techniques 

� Atomic absorption (flame, graphite furnace, cold 

vapor) 

� ICP-OES 

� ICP-MS 

� XRF 

� LIBS 

� Ion Chromatography 

� Flame Emission Spectroscopy 

11/23/2010 

12

Results of Team’s Experiments 

� Dilute-and-shoot (direct dilution) and 

� Closed-vessel digestion provided the best results 

and options for sample preparation procedures 

� ICP-OES and ICP- MS are compendial options for 

analytical determinations 

Elemental Impurities-Procedures 

� When a user does not have a procedure that 

meets the criteria for performance described, 

th then one of f th the referee f procedures d shall h ll be b 

employed. The procedures include: 

� Procedure 1, which can be used for 

elemental impurities generally amendable to 

detection by ICP-OES 

� Procedure 2, which can be used for 

elemental impurities generally amendable to 

detection by ICP-MS. 

11/23/2010 

13

Methodology - Strategy 

� Provide acceptance criteria for methods as 

limits and quantitative tests 

� SSample l preparation ti 

– Solid/neat samples 

– Dilute and shoot 

– Closed-vessel digestion 

� Analysis 

– ICP-OES ICP OES 

– ICP-MS 

� Other methods meeting the acceptance 

criteria are acceptable 

Acceptance Criteria – Limits Test 

� Accuracy – Spiked test samples give 

responses equal to or greater than their 

controls 

� Precision 

– Instrumental methods – RSD for six 

measurements NMT 20% 

– Non-instrumental methods – meet LoD 

requirements 

� SSpecificity ifi it - Th The procedure d must t be b able bl to t 

unequivocally assess each Target Element in 

the presence of components that may be 

expected to be present, including other 

Target Elements and matrix components 

11/23/2010 

14

Elemental Impurities-Procedures 

Quantitative Validation Summary 

Parameter Test 

Accuracy 

Precision 

(Repeatability) 

Comparison of spike sample 

sample 

with standards at 0.5 J, 

1.0J, 1.5J 

Analysis of 6 individual 

sample preps spiked at 1.0J 

Repeatability test performed 

Precision (Intermediate by: separate analyst, 

Precision) different system system, different 

day (only one required) 

Specificity As required 

LOQ, Range and 

Linearity 

Use of Reference Standards 

Acceptance 

Criteria 

80 80-150% 150% recovery 

RSD

Implementation 

� General Notices statement similar to 

Residual Solvents - the standards apply to all 

drug product monographs, even those where 

not specifically mentioned 

� Compendial drug products must comply with the 

limits 

� USP sets standards, regulatory agencies (FDA) 

enforce standards 

– USP does not decide when or if testing is required 

� Proposed official in September, 2013, in line with 

EMEA Guidance 

Stimuli Article-Information 

� Methods for Establishing Exposure Limits 

– EEuropean Medicines M di i Agency A (EMEA) guidance, id 

“Guideline on the Specification Limits for Residues 

of Metal Catalysts or Metal Reagents” (2008) 

– 10g/day dose for drug products for calculation of 

ppm limits 

– 50 kg person for extrapolation from animal data on 

body weight weight-basis basis 

– 70-year lifetime 

– 10% bioavailability for extrapolation from the oral 

permissible daily exposure (PDE) to the parenteral 

PDE 

11/23/2010 

16

Stimuli Article: Elemental Impurities-Comments and 

Responses 

� Topic 1: Instrumental Details 

� Topic 2: Implementation 

�� Topic 3: Specific Metals and Limits 

� Topic 4: Using Residual Solvent Concepts 

� Topic 5: Scope (Dosage Forms, Foods, Dietary 

Supplements) 

� Topic 6: Reference Standards 

� Topic 7: Imminent Threat 

� Topic p 8: Harmonization (EDQM, ( EMEA, MHLW) ) 

� Topic 9: GMP’s and USP 

� Topic 10: Other Comments 

Harmonization 

� ICH is chartering Q3D working group to 

consider elements and limits (not methods) 

– USP and FDA are participants 

– First meeting in June, 2010 

– Second meeting November 7-10, 2010 

� Draft chapters shared with EP and JP 

� Europe has a working party discussing 

additional metals and limits 

� USP potential template for 

harmonization 

� JP has endorsed concepts. Outcome 

on procedures awaits ICH metals and limits 

11/23/2010 

17

USP Website 

� Draft chapter Metals and Limits 

�� Draft chapter Procedures 

� Draft chapter Dietary Supplements 

� Stimuli article – Rationale for Limits 

� Stimuli article - Responses to Comments 

� General Notice statement - Eliminate need to 

reference f in individual monographs 

� Current Expert Panel thinking on draft 

chapters 

USP Next Steps 

� Update draft chapters as needed 

– Any public comment prior to finalizing 

– ICH Q3D recommendations from November meeting 

� Publish new draft chapters for public comment 

– Target publication date – Pharmacopeial Forum 37(2) 

� Note – Starting in 2011, PF free and available to 

all who register 

11/23/2010 

18

Acknowledgements 

Heavy Metals Advisory Panels 

� Nancy y Lewen • Tim Shelbourn 

� Robert Wiens • Assad Kazeminy 

� Steve Dentali • Greg Turk 

� Courtney Callis • Roland Frotschl 

� Mamata De • Bruce Fowler 

� Anna Fan • Richard Ko 

� Chuck Barton • John Kauffman 

USP Staff 

Kahkashan Zaidi, Todd Cecil and Gabriel Giancaspro 

Thank you! 


11/23/2010 

19

SUMMARY OF THE TOXICITY OF METALS 

Bruce A. Fowler Ph.D., A.T.S 

Division of Toxicology and 

Environmental Medicine 

Agency for Toxic Substances and 

Disease Registry 

Atlanta Atlanta, GA 30333 

CONTEMPORARY 

RISK ASSESSMENT ISSUES 

• NEED FOR OBJECTIVE MEASURES (BIOMARKERS) OF CELL 

INJURY FROM TOXIC CHEMICALS THAT MORE PRECISELY LINK 

EXPOSURE (DOSE –ADMINISTERED ADMINISTERED OR MEASURED BY 

CHEMICAL ANALYSIS IN TARGET TISSUES) AND AND MECHANISMS 

OF TOXICITY THAT OCCUR PRIOR TO CLINICAL DISEASE 

• NEED FOR IMPROVED MODE OF ACTION BIOMARKER 

BIOMARKER-BASED BASED 

RISK ASSESSMENTS FOR SENSITVE SUB-POPULATIONS 

SUB POPULATIONS, , 

MIXTURE EXPOSURES AND NEW PRODUCTS (EG. 

NANOMATERIALS) 

• TO BE OF MAXIMAL VALUE, IT IS ESSENTIAL TO ESTABLISH 

MECHANISTIC LINKAGES BETWEEN BIOMARKERS. 

BIOMARKERS. 

MODULATING FACTORS AND OTHER PARAMETERS OF 

TOXICITY 

1

TOXIC METALS / METALLOIDS REPORTED AT 

ELEVATED CONCENTRATIONS IN HEALTH CARE 

PRODUCTS FOR THE PERIOD 1989 – 2008* 

Element Health Care Product Year Cited 

Arsenic Injectables in glass ampoules 2006 

Baby rice products, Ayurvedic herbals 2004 , 2008 

Cadmium Medicinal plants, Nigerian herbals 1989, 2005, 2006 

Lead Pharmaceuticals, Ayurvedic herbals 1989, 2004, 2007 

Mercury Ayurvedic herbals/Herbal medicines 2004, 2007 

Selenium Nigerian herbals, Dietary supplements 2006, 2008 

* Limited review of the open literature using Google and PubMed 

ENVIRONMENTAL STANDARDS FOR TOXIC 

METALS/METALLOIDS 

ATSDR MRL (Chronic) U.S. EPA EXPOSURE STANDARDS 

AIR WATER 

LEAD (Pb) NA 1.5ug/M 3 15ug/L 

CADMIUM (Cd) 0.2 ug/kg/day NA 5ug/L 

ARSENIC (As) 0.3 ug/kg/day NA 10 ug/L 

MERCURY 2.0 ug/kg/day (oral -intermed) 5lbs/24HRS* 2ug/L 

(Hg -inorganic) 0.2ug/kg/day (inhalation) 

SELENIUM (Se) 5 ug/kg/day NA 50ug/L 

* Point Sources 

2

ESTIMATED ADULT DIETARY INTAKES FOR Pb, 

Cd, As, AND Hg (mean ug/day)* 

Males Females 

Pb 14.9 14.8 

Cd 18.5 19.3 

As 50.6 58.5 

Hg 8.2 8.6 

*(Data from MacIntosh, DL et al. Env. Hlth Persp. 104:202 104:202-209, 209, 1996) 1996) 

MAJOR DIETARY SOURCES* OF TOXIC 

ELEMENTS IN THE U.S. FOOD SUPPLY 

METAL / 

METALLOID FOOD SOURCE 

ARSENIC SEAFOOD, RICE PRODUCTS 

CADMIUM SPINACH, SEEDS & GRAINS 

LEAD CHOCOLATE, SWEET PICKLES 

MERCURY FISH 

SELENIUM** FISH, LIVER, PORK, NUTS 

* U.S. FDA DIETARY SURVEY – BASED UPON HIGHEST MEAN CONCENTRATION 

(MG/KG) 

**ALSO AND ESSENTIAL ELEMENT 

3

Chemical 

Exposures 

NHANES III BLOOD VALUES FOR COMMONLY ENCOUNTERED 

METALS AND METALLOIDS 

National Health & Survey Examination, 1999 1999-2002 2002 

Source: Third National Report on Human Exposure to Environmental 

Chemicals (CDC) 

Geometric mean concentration in μg/L g/L for the US population aged 

>6 6 years 

Survey yrs G Mean (95% CI CI) CI CI) 

Arsenic (Total) 03 03-04 04 65.4 (48.7-83.3) (48.7 83.3) urine* 

Cadmium 99-00 99 00 .412 (.378-.449) (.378 .449) blood 

Lead 99 99-00 00 1.66 (1.60-1.72) (1.60 1.72) blood 

01 01-02 02 1.45 (1.39-1.51) 

(1.39 1.51) 

Mercury 99-00 99 00 .343 (.297-.395) (.297 .395) blood 

01 01-02 02 .318 (.268-.377) 

(.268 .377) 

* See Caldwell, KK et al. -J. J. Exp. Env. Epi Epi- 2008 ee-pub. 

pub. 

POPULATIONS AT RISK 

General 

Population 

Increased Risk of Toxicity 

Sensitive 

Subpopulation 

Age 

Gender 

Nutritional Status 

Genetic Susceptibility 

Mixture Exposures 

4

• GENOMICS: 

GENOMICS 

“OMIC” BIOMARKERS 

: CHEMICAL 

CHEMICAL-SPECIFIC SPECIFIC ALTERATIONS 

IN GENE EXPRESSION PATTERNS IN TARGET 

CELL POPULATIONS 

• PROTEOMICS: 

PROTEOMICS 

: CHEMICAL 

CHEMICAL-SPECIFIC SPECIFIC 

ALTERATIONS IN THE ACTUAL EXPRESSION OF 

GENE PRODUCTS (PROTEINS) 

• METABOLOMICS/METABONOMICS: 

METABOLOMICS/METABONOMICS 

: CHEMICAL 

CHEMICAL- 

INDUCED ALTERATIONS IN IN ESSENTIAL 

ESSENTIAL 

BIOLOGICAL PATHWAYS WITH MEASUREMENT 

OF METABOLIC PRODUCTS / PRECURSORS IN 

ACCESSIBLE BODY FLUIDS (EG: URINE) 

BIOMARKER MODIFYING FACTORS 

• DOSE DOSE RESPONSE/TIME COURSE 

• EXPOSED POPULATION (AGE, GENDER, NUTRITIONAL 

STATUS, GENETIC SUSCEPTIBILITY) 

• COMPENSATORY MECHANISMS (INDUCIBLE ENZYME 

SYSTEMS, METAL METAL-BINDING BINDING PROTEINS, STRESS PROTEINS) 

• ANTIOXIDANT SYSTEMS (E.G., GLUTATHIONE) 

5

LEAD EXPOSURE IN THE GENERAL U.S. POPULATION 

COMPARISON OF CURRENT CDC BLOOD LEAD (Pb) LEVEL LEVELOF OF CONCERN 

AND 

AVERAGE BLOOD LEAD MEASURED IN THE U.S. POPULATION BY NHANES III 

*CDC / WHO BLOOD LEAD (Pb) LEVEL OF CONCERN = 10ug/dl 

*NHANES III AVERAGE BLOOD LEAD VALUE = 1.6 ug/dl 

NOTE: GROWING RECOGNITION THAT FOR SOME INDIVIDUALS THERE 

MAY BE BE NO THRESHOLD THRESHOLD FOR LEAD TOXICITY* 

*(See NAS/NRC –”Measuring Lead Exposure in Infants, Children and Other Sensitive Populations”, 

1993) 

7

MECHANISM BASED BASED /MODE OF 

OF 

ACTION BASED RISK 

ASSESSMENTS 

8

AMINO ACID COMPOSITIONS 

KIDNEY PbBP’S 

AA (mole%) DBI Thymosin Rat Monkey 

CYS - - 3 - 

ASP 8 9 13 9 

GLU 9 16 12 10 

LYS 29 25 7 11 

SER 5 11 5 16 

GLY 8 5 8 12 

HIS 7 5 2 - 

ARG 1 - 6 2 

THR 5 2 5 7 

ALA 8 5 5 13 

PRO 1 5 1 9 

TYR - - 5 2 

VAL 2 - 5 5 

MET 2 5 3 2 

ILE 3 5 4 - 

LEU 5 7 8 - 

PHE 3 2 6 2 

9

INCIDENCE OF RENAL 

LEAD INTRANUCLEAR INCLUSION BODIES 

Experimental features: Rats, n = 13 in each experimental group 

Diet - Pb, Cd, Inorganic, Organic As 

EExperimental i t lG Group IIncidence id of fI Inclusions* l i * 

Control 0/13 

Pb 10 /13 

Cd 0 /13 

Inorg As 0 /13 

Org As 0 /13 

Pb x Cd 0 /13 

Pb x Inorg As 10 /13 

Pb x Org As 11 /13 

Cd x Inorg As 0 /13 

Cd x Org As 0 /13 

Pb x Cd x Inorg As 0 /13 

Pb x Cd x Org As 2 /13 

---------------- 

13

POTENIAL SIGNIFICANCE OF LEAD LEAD-BINDING BINDING PROTEINS 

IN HUMAN TISSUES FOR RISK ASSESSMENTS 

• Individuals vary in their susceptibility to Pb poisoning 

• A portion of this variability at lower levels of Pb exposure 

appears t to be b due d in i part tt to genetic ti polymorphisms l hi in i major j 

lead -binding binding proteins in blood (eg. ALAD) which is also a 

sensitive biomarker of lead biological activity in the heme 

biosynthetic pathway. 

• Cellular mechanisms of Pb toxicity are not fully understood 

and may be modulated by a number of molecular mechanisms 

(eg. Pb - binding proteins in target tissues such as kidney and 

brain) 

• No apparent threshold for neurobehavioral toxicity 

14

EMERGING ISSUES 

•INCREASING LEVEL OF CONCERN FOR 

SENSITIVE SUB-POPULATIONS AT LOW DOSE 

METAL EXPOSURE LEVELS 

•IMPORTATION OF PHARMACEUTICAL / 

HERBAL MEDICAL PRODUCTS WITH FEED 

STOCKS FROM DEVELOPING COUNTRIES 

•NEW PRODUCT LINES WITH UNKNOWN 

PROPERTIES AND POTENTIAL CONTAMINANTS 

(EG (EG. HERBAL - BASED NANOMATERIALS) 

•NEED FOR RAPID COST- EFFECTIVE 

SCREENING SYSTEMS CAPABLE OF DETECTING 

CONTAMINATING METALLICS IN HEALTH – 

CARE PRODUCTS 

SUMMARY AND CONCLUSIONS 

*MOA TOXICOLOGY DATA / RISK ASSESSMENTS HAVE 

INCREASED AWARENESS OF POTENTIAL METAL/METALLOID 

TOXICITIES AT LOW DOSE LEVELS 

*INCREASED CONCERN FOR EXPOSURES OF SENSITIVE SUB- 

POPULATIONS TO METALLICS AND METALLIC MIXTURES IN 

NEW FORMS (EG. NANOMATERIALS) 

*GLOBALIZATION HAS RESULTED IN NEW SOURCES AND 

SOMETIMES UNTESTED SOURCES OF FEED STOCKS FOR 

HEALTH CARE PRODUCTS 

*MODERN/HIGHTHROUGH PUT SCIENTIFIC 

* MODERN / HIGH THROUGH –PUT SCIENTIFIC 

APPROACHES FOR SCREENING HEALTH CARE PRODUCTS 

FOR METALLIC CONTAMINANTS ARE INCREASINGLY 

AVAILABLE 

15

Pharmaceutical Applications of Atomic Spectroscopy 

USP Proposed Chapter and 

Techniques for Metals Impurities Control 

2010 USP AAPS Workshop, New Orleans, LA 

Nancy Lewen Lewen, Principal Scientist 

Bristol-Myers Squibb Co. 

R&D, Analytical R&D 

New Brunswick, NJ 

Bristol Bristol-Myers Myers Squibb Company 

Page 1 

Outline 

�Why y look at metals in ppharmaceuticals? 

�What techniques are most commonly 

used? 

– Pros and cons 

�Chapter p 

– Flexibility 

– Accountability 

– Believability 

Bristol Bristol-Myers Myers Squibb Company

Need for testing of Metals in Pharmaceuticals 

• Toxicological information regarding metals has improved over the 

last 100+ years, leading to concerns regarding metals intake, in 

general 

• EMEA Guideline on Residues of Metal Catalysts 

� Only applies to metals that are part of the synthesis 

• Proposed changes to USP , Heavy metals 

� Applies to any extraneous metals, whether part of the process or 

inadvertently added in some way 

� Includes the “Big 4:” Cd, As, Pb, Hg 

� Ultimately applies to drug product 

• Compendial methods do not provide accurate or element-specific 

information regarding metals in pharmaceuticals 

� Modern instrumental techniques provide accurate, element-specific 

information, with much greater sensitivity 


Page 2 

Techniques Commonly Used for Metals 

Analysis 

• Atomic Absorption-based 

�� Flame AA 

� Graphite Furnace AA 

• Plasma-based 

� ICP-AES (ICP-OES) 

� ICP-MS 

• Wet chemistry 

� Hydride generation (can be combined with AA-based or plasmabbased 

d techniques 

t h i 

• Solid analysis techniques 

� XRF, LA-ICP-MS, LIBS 


Advantages and Disadvantages of Flame AA 

� Advantages 

– Good for ppm-% level components or contaminants 

– CCommonly l used d for f Na, N KK, CCa, MMg, FFe, ZZn, Li 

– Inexpensive vs. Plasma-based techniques (Availability in QC 

labs) 

– Ease of use and rapid generation of results 

– Choice of sample preparation/introduction 

� Hydride generation good for As, Se; cold vapor for 

Hg analysis 

� Disadvantages 

– Can use a large amount of sample (up to 4-5 mL/min.) 

– Sensitivity not good for metals with sub-ppm limits (w/w) 

– Potential for interferences 

– Need lamp for each analyte 

– Can only analyze one analyte at-a-time 


Page 3 

ICP-AES (ICP-OES—wavelength-based 

technique) 

• Advantages 

� Rapid (simultaneous, multi-element determinations possible) 

�� Versatile 

� Easily interfaced to various sample introduction strategies 

� Relatively free from matrix interferences vs. AA-based techniques 

� Good sensitivity; ppb-ppm detection easily possible 

� Large linear range (4-5 orders of magnitude) 

• Disadvantages 

� Instrumentation more costly than AAS systems 

� High gas consumption 

� Generally not as sensitive as GFAAS 

� Possible plasma modification needed 

� Need to be mindful of spectral interferences 

� Sometimes requires internal standard 


Torch Configurations 

• Radial (Lateral) 

“Traditional” torch configuration 

Generally more robust than axial configuration 

Can use higher power settings (up to 1750W) 

Generally less sensitive than axial configuration 

Generally less expensive than axial configuration 

• Axial 

Improved sensitivity over radial ICP-AES 

Many elements can be determined at levels 

previously attainable only by GFAAS and/or ICP-MS 

Rapid technique 

FFewer spectral t l interferences i t f than th with ith radial di l ICP ICP-AES AES 

More expensive than radial ICP-AES and less expensive than ICP-MS 

Requires argon for plasma and some kind of sheer gas—usually argon or nitrogen 

Axial plasma employs lower power, resulting in some difficulties when analyzing 

organics. This results in more problems due to carbon buildup. 

• Dual-View 

Option to view plasma either “end on” (axial) or “side on” (radial or lateral) 

Theoretically can take advantage of optimal torch configuration for the analysis 


Page 4 


ICP-MS 

• Advantages 

� Very rapid multi-elemental technique 

� Sensitivity y equivalent q to or ggreater 

than GFAAS 

� Versatile sample introduction 

� Matrix problems not as great as with AA-based techniques 

� Good linear range 

� Can assay halogens 

� Good tool for qualitative or semi-quantitative analysis (Periodic table scan) 

� Nearly an ideal HPLC detector for metal-containing species due to elemental 

specificity 

� Ability to perform isotope dilution--best internal standard 

• Disadvantages 

� Cost: most expensive instrumentation 

� Isobaric and/or matrix interferences for several elements (Fe, Se, Al, As, Si) 

� Not a good technique for C, N, O 

� Considerably more routine maintenance than with other techniques 

� Laboratory requirements to prevent problems with contamination (some labs place 

instruments in a clean room, separate sample preparation area in clean environment) 

� Internal standards required 


Page 5 

Solid Sampling Techniques 

• Laser ablation-ICP-MS (LA-ICP-MS) 

• LLaser-induced i d d breakdown b kd spectroscopy t (LIBS) 

• X-ray fluorescence (XRF) 

• For true quantitation, all need appropriate solid 

standards, which may be difficult to obtain 


LA-ICP-MS 

� Minimal / no sample preparation 

� Small sample size; ability to assay small amount of precious material 

– RRubber bb stoppers t 

– Bulk drug substances 

– Powders pressed into pellets 

– Filters 

� Qualitative analysis (periodic table scan) 

� Ability to analyze layers of samples for spatial distribution of desired 

elements in organs, tumors or other samples 

�� Qualitative results only unless appropriate standards are available 

� Less sensitive than conventional ICP-MS 


Page 6 

Schematic of LA-ICP-MS 

(http://www.gfz-potsdam.de/pb4/pg3/equipment/laicpms.html) 


Laser Induced Breakdown Spectroscopy (LIBS) 

• Well-suited for solid dosage form applications 

• Sample must have at least one metal or “target” analyte 

• Limited availability 

• Considered PAT technology 

• Quantitation possible only with appropriate solid 

standards 


Page 7 

Typical LIBS Instrument (slide courtesy of 

Lydia Breckenridge, BMS) 

Nd:YAG Laser 

(1064 nm, 6-10 ns pulse) 

Pierced Mirror 

Laser-Induced Plasma 

Sample 

Focusing 

Lenses 

Y Axis Title 

Fiber Optic 

400 

350 

300 

250 

200 

150 

100 


50 

0 

407.747 

SSpectrometer t t 

421 421.607 607 

460.829 

-50 

400 410 420 430 440 450 460 

XA i Titl

XRF 

• For true quantitation, appropriate solid standards are 

needed 

• Best-suited for larger sample quantities. 

• Doesn’t work well for light elements 


Page 8 

Proposed Chapter 

• Applies to drug product 

• UUnlike lik EMEA Guideline, G id li includes i l d inadvertent i d t t metal t l 

contamination 

• Risk-based approach acceptable 

• Summation or individual element compliance with 

standard 

• Proposed chapter provides analytical information 


Proposed Chapter 

• Companion chapter provides limits and elements 

� Because of specified limits, ICP-OES and ICP-MS provide best options 

for rapid, accurate, multi-element analysis 

• Analysts free to use any method they deem appropriate 

• “Referee” methods available, if analysts do not have method to use 

� Prior to using one of the compendial procedures, must verify its 

appropriateness for a given sample and technique 

– Validation for limit procedures 

� Control sample—reference material for element of interest at 

target g concentration 

� Test sample 1—sample spiked to target concentration in 

triplicate 

� Test sample 2—sample spiked to 90% of target concentration in 

triplicate 

� RSD NMT 20% (n=6) 

� Specificity—no false positives or false negatives 


Page 9 

Proposed Chapter (continued) 

– Validation for quantitative procedures 

� Accuracy 

� Control Samples: 50-150% of limit value for element of 

interest (in triplicate) 

� Test Samples: samples spiked to 50-150% of limit value for 

element of interest (in triplicate)—spike recovery 50-150% 

for mean values 

� Precision 

� RSD NMT 20% (n=6) for spiked samples 

� Intermediate precision: either on different days; OR with 

different instrumentation; OR with different analysts (RSD 

NMT 25%) 

� Specificity 

� No false positives or false negatives 


Procedures 1 and 2 (ICP-OES and ICP-MS, 

respectively) 

• Standards: 2xlimit for a given element and 0.1xlimit for a 

given element 

• System suitability solution: 1 ppm of elements of 

interest 

• All solutions matrix-matched 

• Follow validation requirements 

• Follow manufacturer’s suggestions for program and 

wavelength or m/z. 

• Calculate results based on original sample size 

• Weigh all liquid samples 


Page 10 

What about Wet Chemical Methods? 

• Some non-instrumental techniques may not be able to 

meet the limits for a given element and/or sample 

• Limits selected were driven by toxicological information 

and developed by a team of toxicologists 

• Key is to meet validation requirements 

• Demonstrate that whatever method you chose is 

appropriate for its intended use 


What’s Next? 

• USP to publish revised chapters and 

• IImplementation l t ti timeline ti li to t be b established t bli h d 

• Industry should already be in the process of evaluating 

their raw materials, excipients, API’s and/or drug 

products 

• Provide feedback to USP 


Page 11

Regulating Elemental Impurities 

in Pharmaceutical Products 

Mamata De, Ph.D. 

1

Introduction 

Contents 

Toxicity of Different Metals 

Specification for Elemental Impurities 

Points to Consider 

Current Status 

2

Introduction 

Impurities are unwanted chemicals which may 

affect efficacy and safety of the pharmaceutical 

products 

As per the US Federal Register (Vol. 65, No. 

251): Any component of the new ‘Drug 

Substance’ (Active Pharmaceutical Ingredients- 

API) or ‘drug drug product’ product (Finished dose) that is not 

the chemical entity or an excipient defined as 

impurity 

3

Types of Inorganic Impurities 

4

Specification of Heavy Metals 

Heavy metals have been monitored in 

APIs for many yyyears 

– Some are toxic 

– Some are not toxic but reflect quality issues 

5

Controls on Heavy Metals (HM): USP NF 

of USP/NFF 

Monograpphs 

with 

Speciffic 

Limit 

Number 

350 

300 

250 

200 

150 

100 

50 

0 

Drug Substance 

Drug Product 

Excipient 

0.2 0.3 1 2 5 10 13 15 20 25 30 40 50 60 83 100 

Limit on Heavy Metals (ppm) 

6

Controls on Heavy Metals (HM) & Lead: US/NF 

Numberr 

of USP/NFF 

Monograpphs 

with 

Speciffic 

Limit 

25 

20 

15 

10 

5 

0 

Drug Substance 

Excipient 

2 3 5 10 15 20 25 30 40 50 

Limit on Lead (ppm) 

7

Controls on Heavy Metals (HM) & Lead: US/NF 

• Limits on HM or Pb exist predominantly for the 

components of DP, not the DP themselves 

• 47% of DS, 54% of excipients, and 4% of DP have a 

limit on HM 

- Only 2% of DS and 16% of excipients have a limit 

on Pb 

- Some have limits on HM and Pb 

8

Controls on Heavy Metals (HM): USP NF 

About 4300 monographs 

• 1331 for DS 

619 have a limit on HM 

22 have a limit for Pb 

• 374 for excipients (NF) 


60 have a limit for Pb 

• 2452 for DP 


8 has a limit for Pb 

9

EEvaluation l ti of f Acceptable A t bl Exposure E for f 

Metals in Food and Drugs 

• Human (preferred if good-quality data are available) and animal 

toxicity data associated with exposure to the metal 

• Likelihood of presence of the metal in the article to be tested 

• Level and pattern of use or consumption of the article or product 

• Level of exposure to the metal 

• Other sources of exposure to the metal 

• Other factors that may affect toxicity (e.g., co-exposure to other 

metals) 

• DData quality li and d iindividual di id l variability i bili 

• Special populations at increased risk for toxicity 

10

Quantifying Toxicity 

• Non-cancer effects 

– Impact the development, size, or functioning 

of the whole body or body specific organs, but 

does not lead to the development of 

malignant cells cells. 

– “Toxicity threshold” represents the dose below 

which adverse health effects are not expected p 

to occur. 

– Potential for adverse effects increases as 

ddose increases i above b ttoxicity i it th threshold. 

h ld 

11

Dose Levels 

– NOEL no no-observed observed effect level 

– NOAEL no-observed-adverse effect level 

– LOAEL lowest lowest-observed-adverse observed adverse effect 

level 

– MTD maximum tolerated dose 

–LD 50 

dose which kills 50% of 

population 

12

Dose Estimate of Toxic Effects 

LD50 

• NOAEL -- No observed adverse effect level; highest data 

point at which no observed adverse/toxic effect. 

• LOAEL -- Low observed adverse effect level; lowest point at 

which an observed effect. 

13

Reference Dose (RfD) 

�An estimate of the daily dose of a 

chemical that will avoid toxic effects other 

than cancer 

� NOAEL or LOAEL is adjusted by 

uncertainty factors (UF) to allow for 

differences in sensitivity to chemicals 

• HHuman ddata: UF = 10 

• Animal data: 

UF = 100 (NOAEL) (NOAEL), 1000 (LOAEL) (LOAEL), 1000 

(NOAEL, less data) 

14

RReference f D Dose (RfD) ( (cont.) t ) 

� RfD = NOAEL/UF 

Ex. 100 µg/kg/day (NOAEL) / 100 (UF) = 1 µg /kg/day 

(RfD) 

� Use RfD to establish allowed concentrations 

Ex Ex. Based on 10 g of drug product taken per day day, a PDE 

of 0.1 µg per gram (parts per million; ppm) is derived 

15

Dose-Response Curves (non-carcinogens) 

Response 

% 

RfD NOAEL LOAEL 

Dose (mg/kg-day) 

16

Migration of Lead into Food or Drugs from Containers, 

Cookware, or Processing Equipment 

Lead glazed ceramics-Mexico 

Candy wrappers 

Decorations on glassware g 

Cocoa and chocolate 

Wine - especially certain imports 

Lead solder or joints in eq equipment ipment - OK dialysis dial sis incident 

17

Massachusetts Bay Law Law, September 33, 

1723; AN ACT FOR PREVENTING ABUSES IN 

DISTILLING S G OF O RUM U AND OTHER O STRONG S O G LIQUORS QUO S 

WITH LEADEN HEADS OR PIPES 

• Whereas the strong liquors and spirits that are distilled through 

leaden heads or pipes are judged on good grounds to be 

unwholesome and hurtful; notwithstanding which some persons to 

save charge may be led into the making or using of such heads, 

worms or pipes; for remedy and prevention whereof, --- 

• “Be it enacted by the Lieut. Governor, Council and Representatives 

of Gen’l Court assembled, and by the authority of the same, 

• “(Sect (Sect 1) That no person whatsoever shall make use of any such 

leaden heads or worms, for the future, and that whosoever shall 

presume to distil, or draw off any spirits or strong liquors thro’ such 

leaden heads or worms; upon legal conviction thereof before any of 

his majesties courts of records records, shall forfeit and pay a fine of one 

hundred pounds.....” 

18

DDeliberate lib t Ad Adulteration lt ti or SSubstitution b tit ti 

In Hungary in 1994, hundreds of people developed acute 

lead poisoning, some with PbB reaching 200 µg/dL, after 

consumption of paprika illicitly laced with lead tetroxide, 

a red colored lead salt, that brightened the spice’s spice s color 

and increased its weight. 

A one month national ban on paprika sales caused 

massive i public bli reaction; i 44 people l were arrested, d and d 

over 3300 tons of paprika were seized and destroyed. 

19

Lead as impurities in food or drug 

Vitamins and supplements pp - especially p y minerals 

Summer 1997: TLC trial of pediatric lead chelation voluntarily 

recalled vitamin/mineral supplement pp ggiven to all pparticipants p 

because of 32 µg Pb/tablet 

Background dietary lead for children: median 2 µg/day 

Source traced to newly sourced iron ingredient used in 

compounding containing 250 - 600 ppm Pb 

20

Analysis of dietary supplements for arsenic, 

cadmium, mercury, and lead using inductively 

coupled l d plasma l mass spectrometry t t [Dolan et al. J 

Agric Food Chem 2003; 51:1307-1312] 

CFSAN team measured selected elements in 95 

predominantly botanical or herbal dietary supplements 

purchased h d iin WWashington hi t DC area iin 1999 

Levels of lead in 11 products p ( (12%) ) yyielded daily y lead 

doses in excess of “provisional tolerable intake” values 

for children 

Dose range for high Pb products 7.6 - 486 µg Pb/day 

21

Lead in pharmaceutical products and dietary 

supplements [Kauffman et al. Reg Toxicol Pharmacol 48:128- 

134; 2007] 

• CDER study of Pb concentration in 45 common prescription and 

OTC drugs and products 

• Mean Pb concentration = 0.048 ppm (range 0 to 0.5). Avg. daily 

dose of Pb at maximum recc. intake = 0.22 µg/day 

• 5 products yielded daily Pb dose > 1 µg/day when 

consumed at maximum daily dose: 

» Turns [sic] Chewable Tablets 2.67 µg/d 

» Oyster Shell Calcium (CVS) 1.66 

» Pepto Bismol 1.59 

» Commit (smoking cessation) 1.02 

» Ibuprofen/APAP (India) 1.07 

22

Blood Lead Levels less than 5 µg/dL Associated with Lower 

Scores in Math and Reading in School Children 

[ Lanphear et al. Public Health Reports. 115:521-9; 2000 ] 

NHANES III data-set; n = 4853 children ages 6 - 16 years 

Geometric mean BLL = 1.9 µg/dL; only 2% with BLL ³ 10 

BLL Quartile Arithmetic Score* Reading Score* 

² 1 µg/dL 95.8 94.5 

1.1 - 1.9 µg/dL 94.0 93.3 

20 2.0 - 30 3.0 µg/dL /dL 94 94.7 7 93 93.0 0 

> 3.0 µg/dL 91.4 88.2 

* WRAT. Adjusted for gender, race/ethnicity, Poverty Index Ratio, 

parent education, ferritin, cotinine. Quartiles differ P < 0.0001 

23

The Relationship Between Blood Lead and Blood 

Pressure in the NHANES II Survey 

Schwartz J Environ Health Persp. 78:15-22; 1988 

• Representative Cross sectional survey of US population 

20,322 persons examined 

• Mean blood lead in adults 13.1 mcg/dl (12.7-13.7) 

• Blood lead significantly associated with systolic and 

diastolic blood pressure, after controlling for age, BMI, 

demographic, g p multiple p 

nutritional factors 

24

[ Pirkle et al, 1985 ] 

25

Acute 

• GI effects 

– colic, severe pain 

– severe constipation 

• Acute 

encephalopathy 

• Acute nephropathy 

Children 

• Growth retardation 

• Behavioral 

Lead Toxicity 

Chronic 

• Peripheral, central 

neuropathy th 

• Cardiac toxicity 

• Ch Chronic i nephropathy h th 

• Saturnine gout 

• Reproductive effects 

• Hypertension 

• Anemia 

26

Lead RfD 

• NOAEL = Lead is a no threshold toxin; therefore, there is 

no RfD 

• In 1994 FDA adopted an allowable level for lead at 5 ppb 

as a bottled water quality standard regulation (59 FR 

26933). 

• AAssuming i an average consumption ti of f 2 L/d L/day of f the th 

bottled water, the oral PDE is 10 µg/day for a 50-kg 

person 

• Based on 10 g of drug product taken per day, a PDE of 1 

µg per gram (parts per million; ppm) is derived 

• Anticipating p g that the p parenteral PDE will be 1/10 of oral, , 

0.1 ppm is derived 

27

Elemental Mercury 

Found in mercury thermometers 

Used in the extraction of gold 

from ores 

Up to 6,000 tons/yr released 

naturally from earth’s crust 

2,000-3,000 tons/yr from 

released from anthropogenic 

sources 

28

Th Three Forms F of f Mercury M 

Elemental, Inorganic, Organic 

• Hgo (elemental mercury) 

• Hg+ g ( (mercurous inorganic g mercury) y) 

• Hg++ (mercuric inorganic mercury) 

• Methyl y mercury y and dimethylmercury y y ( (organic g 

forms) 

• Only y expect p inorganic g ppossibly y in 

pharmaceuticals 

29

How can Elemental Mercury 

Enter the Body? 

Oral Dermal Inhalation 

30

Mercury: Toxicity 

• Methyl y mercury y is the form of mercury y that is most 

likely to cause adverse health effects in the general 

population 

• Microorganisms in the environment can convert 

inorganic mercury to the organic form methyl 

mercury. This form can build up in the environment 

and d accumulate l t iin certain t i ffreshwater h t and d saltwater lt t 

fish, and marine mammals 

• Methyl mercury is readily absorbed absorbed, distributed in 

nervous system, kidney, and GI tract, an oxidation 

reduction cycle is involved in its metabolism, 

elimination is slow (half life >35 days) days), excreted 

mainly via urine 

31

Inorganic Mercury Poisoning 

• Gastrointestinal phase: Hg2+ is a potent GI irritant 

– gingivitis, stomatitis 

- oesophageal oesophageal, gastric gastric, small and large bowel erosions 

- haematemesis, bloody diarrhoea, CVS collapse 

• Systemic toxicity: Hg2+ y y g inhibits sulphydryl p y y enzymes y 

– hypotension, lactic acidosis 

N h t i it H 2+ • Nephrotoxicity: Hg d it i th t b l ATN 

2+ deposits in the tubules → ATN 

– acute renal failure 

- potentially leads to CRF in survivors 

32

Mercury RfD 

• The presence p of methyl y mercury y in ppharmaceutical 

products is extremely unlikely; therefore, based on 

mercuric chloride 

• RfD was based on formation of mercuric-mercuryinduced 

autoimmune glomerulonephritis 

• RfD = 0.3 µg/kg/day or 15 µg/day for a 50-kg person 

• Based on 10 g of drug product taken per day day, a PDE of 

1.5 µg per gram [parts per million (ppm)] is derived 

• AAnticipating ti i ti that th t the th parenteral t l PDE will ill bbe 1/10 of f oral, l 

0.15 ppm is derived 

33

A 27 y.o. male resident of Ramnigar 

village in West Bengal Bengal, India 

developed the gradual onset of 

spotted hyperpigmentation 10 years 

earlier. Until one year y ago, g , he 

experienced dysesthesias of the 

palms and soles. 

Physical exam: 

Diffuse fine-freckled 

hyperpigmentation 

HHyperkeratoses k t of f the th palms l and d 

soles 

Left basilar lung field crepitance 

34

• NOAEL = 0.8 µg/kg/day 

Arsenic RfD 

• LOAEL = 14 µg/kg/day for hyperpigmentation, keratosis, and possible 

vascular complications 

• UF = 3 (human data) 

• OOral l RfD = 00.3 3 µg/kg/day /k /d 

• 50 kg person = 15 µg oral per day 

• Based on 10 g of drug product taken per day, a PDE of 1.5 µg per gram 

(parts per million; ppm) is derived 

• Anticipating that the parenteral PDE will be 1/10 of oral oral, 00.15 15 ppm is derived 

36

Itai Itai-Itai Itai or “ouch “ouch-ouch” ouch” disease 

37

Target Organs of Itai-Itai Disease 

Kidney 

38

• NOAEL = 5 µg/kg/day 

• UF = 10 (intrahuman variability) 

• Oral RfD = 0.5 µg/kg/day 

Cadmium RfD 

• 50 kg person = 25 µg oral per day 

• Based on 10 g of drug product taken per day, a PDE of 2.5 µg per 

gram ga (patspe (parts per million; o ;pp ppm) ) is sde derived ed 

• Anticipating that the parenteral PDE will be 1/10 of oral, 0.25 ppm is 

derived 

39

• Platinum 

• Iridium 

• Rhodium 

• Osmium 

• Ruthenium 

• Palladium 

• Thalium 

Toxicity of Trace Metals 

• Antimony 

• Barium 

• Beryllium 

• Vanadium 

• Copper (Cu II) 

• Nickel 

• Molydenum 

40

Proposed Limits (Oral) 

Elements USP/PDE/ EPA/SFDW EMEA 

(µg/day) µg/L 

Lead 10 15 None 

CCadmium d i 5 5 NNone 

Arsenic 15 10 None 

Mercury 15 15 None 

Palladium 100 None 100 

Platinum 100 None 100 

Iridium 100 None 100 

Rhodium 100 None 100 

Ruthenium 100 None 100 

Osmium 100 none 100 

41

Proposed Limits (Oral) 

Elements USP/PDE EPA/SFDW EMEA 

µg/day µg/L µg/day 

Chromium 250 0.1 250 

Copper 2500 13 1.3 2500 

Molybdenum 250 None 250 

Nickel 250 None 250 

Vanadium 250 None 250 

Manganese 2500 None 2500 

IIron NNone NNone 13000 

Zinc None None 13000 

Antimony 0.006 

Barium 2 

42

Proposed Limits (O (Oral) ) 

Elements USP/PDE EPA/SFDW EMEA 

µg/day /d 

µg/L µg/day 

Beryllium None 0.004 None 

Selenium None 005 0.05 None 

Thallium None 0.002 None 

Cobalt None None None 

Aluminum None None None 

Indium None None None 

Tin None None None 

Tungsten None None None 

Strontium None None None 

Lithium None None None 

Boron None None None 

43

Points to consider: What metals to monitor? 

Toxicity of potential target metals 

Toxicity of individual metals 

Toxicity of combined groups of metals 

Potential target organs 

What if individual metals are not terribly toxic toxic, but 

more than one has an impact on the same target 

organ? 

Concerns: Pb, Cd 

44

Points to consider: What concentration limits are 

required? 

Depends on patient population 

Depends on daily dosage 

Depends on type of dosage form 

Depends on whether it’s for an acute or a chronic 

condition 

Depends epe ds on o metal eta 

45

CCurrent S Status/Recommendation/s 

/ / 

ICH is discussing the topic 

For setting specification for arsenic, lead, cadmium, and mercury 

following USP's specification published in Pharmaceutical Research: 

Volume 27, Issue 5 (2010), Page 750. 

http://www.springerlink.com/openurl.asp?genre=article&id=doi:10.1007/ 

s11095-010-0080-3. 

FFor setting tti specification ifi ti ffor th the ttrace metal t l iimpurities, iti EMEA’ EMEA’s 

Guidance, 2009 is worthwhile for consistency and transparency 

Consulting g EPA’s SFDW document for water soluble metals might g be 

helpful 

Document entitled ‘Safe upper levels for vitamins and minerals’ 

published by Expert Group on Vitamins and Minerals’ Minerals might be another 

resource to consult for recommending safe limit for some of the metals 

46

Current and Future Directions and 

Implications for Complex Biologics 

AAnita it Szajek, S j k Ph Ph.D. D and d Ti Tina MMorris, i Ph Ph.D. D 

A USP Sponsored Workshop at 2010 AAPS-PSWC Meeting 

Impurities, Adulteration, and the Changing Role of the USP in Global Drug Quality 

November 13-14, 2010 

Morial Convention Center, New Orleans, LA 

Outline 

1. The traditional and new role of the Pharmacopeia 

2. Challenges associated with development of biological 

standards 

3. USP’s B&B program 

� The role of vertical and horizontal standards 

� Examples of Definition, Identification and Potency 

4. What can we expect in the future? 

� Product class chapter approach 

11/29/2010 

1

The Pharmacopeia of the United States of America 

�1820 

� The First Edition of USP 

�“In the United States the evil of irregularity and 

uncertainty in the preparation of medicines has 

been felt with peculiar weight…” 

The Changing Role of Pharmacopeia 

1820 

• To ensure accurate 

dispensation of medicines by 

pharmacists 

p 

Example monographs 

• Decoction of Seneca 

Snakeroot 

• Extract of Peruvian Bark 

• Syrup of Buckhorn 

• Purified Mercury 

Now 

• To provide public standards, 

specifications, and test methods 

for quality control for APIs APIs, 

excipients, and drug products 

Modern Day Monographs 

• Insulin Human 

• Sipuleucel-T 

• Cryopreserved Human 

Fibroblast-Derived Dermal 

Substitute 

11/29/2010 

2

Compendial Challenges for Biological Standards 

• Globalization means the exchange of goods and 

services worldwide 

– When developing test methods, different sources of APIs must be 

considered 

– Many biologics are still human- or animal-derived 

• Test methods must be sufficient and robust, based on 

technologies that are state-of-the-art and available 

globally at reasonable costs 

– Biologics are often very complex, requiring highly specialized 

characterization technologies 

• To stay current and useful, pharmacopeias must follow 

developments in regulatory, medical practice and 

technologies, but stay flexible 

– ICH guidelines, quality risk management, quality systems, PAT, 

biosimilars 

Compendial Standards Continuum in the Biopharmaceutical 

Lifecycle 

Early IND Clinical BLA/NDA Market 

USP Horizontal Standards: General Chapters & 

Reference Materials for Procedures 

USP Horizontal Standards: General Chapters & 

Reference Materials for Ancillary & Process Materials 

Pending 

Standards 

USP Vertical Standards: 

Monographs and Reference 

Materials 

11/29/2010 

3

Building Blocks of USP Biological Standards 

Horizontal Standards 

Biotechnology-Derived Articles— 

Peptide Mapping 


Isoelectric Focusing 


Polyacrylamide Gel Electrophoresis 


Total Protein Assay 

Chromatography 

Microbial Enumeration Tests 

Bacterial Endotoxins Test 

USP Reference Standards 

Vertical Standards 

Filgrastim 

monograph 

Vertical Standards & Key Quality Attributes 

Benefits: 

• Clearly define a product’s identity, strength and purity, as well as 

other important p qquality y attributes 

• Allow independent testing and verification based on a public 

standard 

Challenges in Standard Development: 

• Inclusion and bridging to new analytical technology 

• Complexity of specifications and system suitability criteria 

• Product- vs. Product-class specific standards 

• Bi Biological l i l potency assignments i and d unit i maintenance i 

– Across manufacturers 

– Internationally 

11/29/2010 

4

Definition – key elements 

• What is the chemical composition? 

• How can it be made? 

– Key process-related requirements may appear here, e.g. viral 

inactivation, species origin, etc. 

• What activity or activities does it have? 

• Is there a minimal 

– Activity 

– Ratio or content of a moiety y or specific p chemical ggroup p 

Elements of Identification for Biological Products 

• Orthogonality 

– More than one test should be used to demonstrate identity, each 

test should measure a different attribute of the molecule 

• Specificity – see USP Validation of Compendial 

Procedures and ICHQ2R1 

– Identification Tests require the demonstration of specificity as the 

primary goal in validation 

• Activity/function 

– May be called out separately as Bioidentity 

– May also be part of the Definition 

11/29/2010 

5

Identification – Example 1: Insulin Human 

Identification— 

• A: The retention time of the major peak in the 

chromatogram of the Assay preparation corresponds to 

that in the chromatogram of the Standard preparation, as 

obtained in the Assay. 

• B: Determine the peptide fragments, using the following 

peptide mapping procedure. 

Identification and other Tests are often 

linked 

Identification – Example 2: Heparin Sodium 

Identification 

• A: 1 • A: H NMR Spectrum 

1H NMR Spectrum 

• B: Chromatographic Identity 

• C: Anti-Factor Xa to Anti-Factor IIa Ratio 

• D: Identification Tests – General: Sodium 

11/29/2010 

6

Identification and the “Absence of” Concept 

In Heparin Sodium: 

1 H NMR Specification for Identity of Heparin 

H1 of GlcNAcc/GlcNS, 

6S 

H1 of o IdoA2S 

1 2 

�H 

OD 

ppm 

H2 of o GlcNS 

3 

4 

of GlcNAc 

�TSP 

8.00 

Acceptance Criteria 

•No unidentified signals greater than 4% of the mean of signal height of 1 and 2 are 

present 

in the following ranges: 0.10-2.00, 2.10-3.20, and 5.70-8.00 ppm. 

•No signals greater than 200% signal height of the mean of the signal height of 1 and 2 

are 

present in the 3.35-4.55 ppm for porcine heparin. 

Methyl 

0 

11/29/2010 

7

Assay – Amount of Substance or Activity? 

ICH Q6B: 

The Measurement Challenges 

• What is the potency of a multi-component mixture, i.e. 

which activity or activities do we measure? 

– Example: Pancreatin has lipase, lipase protease, protease and amylase 

activities 

• Should an assay be specific to one activity or broadly 

capture multiple attributes? 

– Example: Heparin potency assessment by plasma clotting or 

specific aIIa and aXa assays 

• Should activity be measured directly (absolute) or 

comparatively (vs (vs. a standard?) 

• What is the influence of matrix effects and how can they 

be controlled? 

11/29/2010 

8

Strategy for B&B Product Classes 

Monograph 

Product Class Overview: Information 

Common Product Class Quality 

Attributes 

Analytical 

Procedures 

Ancillary 

Materials 

Monograph Monograph Monograph Monograph 

Product Class Chapter – Scope and Purpose 

� Definition of critical quality attributes common to a product 

class: 

Establish a “pick list” of tests suitable and necessary to establish 

quality, strength and purity across the product class, e.g.: 

� Measure and define common post-translational modifications, e.g. 

glycosylation in conserved regions of MAb structure 

� Define tests and acceptance criteria for common product-related 

impurities or degradants 

– Deamidation or oxidation 

– Aggregates 

– RResiduals id l of f common process or ancillary ill materials: t i l protein t i AA, nucleotidic l tidi 

impurities, host cell protein 

� Establish accepted assay approaches to activity and potency 

determination, e.g. enzyme measurement and unit establishment 

� Link to validated and public compendial procedures that apply 

broadly to the entire product class 

11/29/2010 

9

Conclusions 

• A pharmacopeial monograph captures the key quality 

attributes of a medicinal product in terms of identity, 

strength and purity 

• For biological medicines key quality attributes are often 

more difficult to define and require multiple, orthogonal 

tests 

• By focusing on and grouping key quality attributes of a 

class of drug products, a pharmacopeial monograph is 

able to accommodate complex and multi multi-manufacturer 

manufacturer 

products 

11/29/2010 

10

11/29/2010 

11

Characterisation of Heparin; Recent 

Responses to Contamination Events 

Barbara Mulloy, NIBSC 

National Institute for Biological Standards and Control 

Assuring the quality of biological medicines 

Overview 

• Background: Heparin and the OSCS contamination problem 

• The USP response: a three‐stage approach to rapid but 

thorough monograph revision 

• Answering some questions about the contaminant(s) 

11/29/2010 

1

Clinical uses of heparin 

• Treatment of deep venous thrombosis 

• Prophylactic prevention of postoperative venous thrombosis. 

• Initial prophylactic prevention of thrombosis following a 

myocardial infarct 

• In IV dialysis dialysis to prevent thrombosis in the pumps 

• DIC (disseminated intravascular coagulation) –to prevent 

coagulation and consequent depletion of clotting factors in 

some disorders 

Heparin history 

• 1916: heparin isolated from liver by Jay McLean, a graduate 

student of William H. Howell at the University of Toronto 

• 1935: First human trial of heparin for the prevention of post‐ 

operative thrombosis 

• 1960s‐1970s –main structural features of heparin elucidated 

• 1976 –Low Molecular Weight Heparin invented 

• 1980 – structural basis for anticoagulant activity discovered 

11/29/2010 

2

Heparin structure 

• Heparin is a complex polysaccharide, not a peptide. 

- 

CH2OSO3 * - 

H 

CH2OSO3 H 

H 

O 

O OH 

* * H 

H H * O 

CH2OSO3- COOH H 

COOH 

* - 

H 

OSO H 

O 

O OH 

H 

3 

O 

H 

H H 

O 

H 

H 

H 

- 

- 

H H NH H OSO 

2SO3 3 

OH 

OH 

O 

H 

O 

H 

H 

O 

- 

NH2SO3 H NHR H OH 

R COCH or SO - 

H 

H 

H 

H 

O 

O 

H 

COOH 

O OH 

O 

H 

COOH 

H 

OH 

H 

H 

- 

H OSO3 OH 

H H 

H 

NHR H OH 

R = COCH3 or SO3 

OSO 3 - 

What is the basis for heparin’s 

anticoagulant/antithrombotic actions? 

H 

Heparin is made up of alternating glucosamine and 

uronic acid residues, heavily substituted with 

sulphate 

• The dominant mechanism by which heparin affects coagulation 

is through its interaction with the plasma protein antithrombin, 

a serine protease inhibitor. 

CH 2OSO3 - 

• Antithrombin binds selectively to the pentasaccharide sequence 

shown in the previous slide 

• The two major targets for antithrombin are Factor Xa, which 

converts prothrombin to thrombin, and thrombin itself (Factor 

IIa) which converts fibrinogen to fibrin 

O H 

H 

O 

- 

NH2SO3 11/29/2010 

3

What was the contamination 

problem? 

In early 2008, a cluster of serious adverse events, including 

deaths, was reported by US FDA 

These events followed administration of Heparin Sodium to 

patients. Further incidents were reported in Europe. 

It was rapidly determined that the products involved were 

contaminated with Over‐Sulphated Chondroitin Sulphate 

(OSCS). 

Regulatory g yAuthorities 

and Medicines Control Labs took 

numerous actions: 

Recalls, Market Surveillance, Method Development, 

Monograph Revision (USP, EP, JP). 

What is OSCS? 

• Chondroitin sulphate (CS) is an abundant polysaccharide in the 

extracellular matrix of animals, and occurs in the same tissues 

used to make heparin 

• Like heparin it consists of alternating uronic acid and 

aminosugar monosaccharides, but galactosamine, not 

glucosamine, is the amino‐sugar found in CS 

OSCS is chondroitin sulphate which has been 

treated to increase its sulphate content 

11/29/2010 

4

Why is it dangerous? 

• The mechanisms involved in adverse reactions to OSCS are not 

well understood, but recent publications* publications indicate something 

like the following: 

• The severe reactions to OSCS‐contaminated heparin in patients 

included refractory hypotension 

• OSCS increases activation of pre‐kallikrein to kallikrein in human 

plasma 

• This results in the production of bradykinin bradykinin, which causes 

hypotension 

* for example, McKee et al. (2010) J. Clin. Pharmacol. 50, 1159‐1170. 

Timeline for USP response 

• April‐June 2008: USP completed Stage 1 monograph revision; 

method validation completed 

• June 18 2008: USP publishes Revision Bulletins for Heparin 

Sodium and Heparin Calcium 

• August‐December 2008: USP completed Stage 2 monograph 

revision 

• February 4, 2009: USP posts proposed Heparin Sodium 

monograph on the web 

• March‐April 2009: PF 35(2) Heparin Sodium IRA 

• October 1, 2009: Heparin Sodium IRA becomes official (Stage 2) 

• Jan 2010‐Present: Stage 3 revision in progress 

11/29/2010 

5

Potential characterization methods for 

heparin identity/contaminant detection 

• NMR spectroscopy ( 1H, 13C, 1D, 2D) 

• Capillary electrophoresis 

• Anion‐exchange chromatography 

• ‘Flat’ electrophoresis techniques (agarose, cellulose acetate) 

• Optical rotation 

• Galactosamine in total hexosamine 

• Gel permeation p chromatography g p y 

USP/European Pharmacopoeia (EP) chosen 

methods 

Specific 

Activity 

EP USP 

Not less than 180 IU/mg for EP, not less than 180 USP U/mg � � 

Identification Delays clotting of recalcified citrated sheep plasma � X 

Nuclear magnetic resonance spectroscopy (USP also use as 

impurity test for OSCS) 

� � 

SAX-HPLC also used as impurity test � � 

Anti-Xa to anti-IIa ratio X � 

Impurities Galactosamine in total hexosamine X �; NMT 1% 

Assay Potency �, sheep 

plasma 

clot-based 

�, anti-IIa 

chromogenic 

11/29/2010 

6

NMR spectroscopy – 1D 

1D 1 H NMR can identify and quantify OSCS in heparin. 

Sample 0100‐D01 

Anion exchange chromatography 

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 

A: Strong anion exchange chromatography 

of a contaminated heparin sample before 

and after treatment with nitrous acid 

B: As A, with an overlaid chromatogram of 

extracted d OSCS 

Red = sample 

black = nitrous degraded 

blue = OSCS 

11/29/2010 

7

Orthogonal methods 

• Orthogonal methods provide complementary information about 

the sample under analysis 

• Strong anion exchange chromatography indicates the presence 

of a substance which is more highly sulphated than heparin 

• NMR spectroscopy identifies the presence of OSCS 

• Galactosamine content indicates the presence of any 

chondroitin or dermatan sulphate impurity, whether highly 

sulphated or not 

• The same set of methods would also be useful to find a highly 

sulphated contaminant that was not OSCS 

Anticoagulant assays and specifications ‐ old 

and new 

• Until 2009, the method for assay of potency in the USP and in 

the EP was based on heparin’s heparin s ability to delay the clotting of 

sheep plasma 

• The minimum specific activity for heparin in the USP was 140 

USP units/milligram and in the EP was 150 I.U. per milligram 

• In 2009 2009, the USP potency assay was changed, changed and is now an 

anti‐IIa assay using purified reagents 

• At the same time, the specification was raised to 180 USP units 

per milligram 

11/29/2010 

8

Specific anticoagulant activities, by several assay methods, 

of OSCS, one pure heparin, and two contaminated heparins 

Batch EP Sheep Human 

APTT APTT 

IU/mg IU/mg 

Sheep/Human 

Ratio 

Anti-IIa 

IU/mg 

Anti-Xa 

IU/mg 

%OSCS 

0030-D01 160 145 1.10 146 136 14.5 

0099-D01 175 141 1.24 137 140 29.4 

OSCS 

from 0099 

138 49 2.81 8 12 100 

97/580 233 234 1.00 235 238 0 

Assays using purified reagents, together with an increased specification, 

tend to make the adulteration of heparin with OSCS or similar compounds 

economically pointless 

Stage 3 Revision Requests from FDA 

• FDA would like the proton NMR procedure to have an increased 

sensitivity and detect OSCS down to 0.1% 

• FDA would like to enhance protein and nucleic acid detection 

procedures, reducing the detection limit from 1.0% to 0.1% 

• FDA has requested the addition of a molecular weight 

procedure. It is thought that molecules with very high average 

molecular weights can contribute to heparin induced 

thrombocytopenia 

• FDA would like to add a lipid detection procedure to address the 

presence of fatty acids, triglycerides, and phospholipids in 

heparin 

11/29/2010 

9

OSCS contaminated heparin 

• How did heparin become contaminated with OSCS? 

• Was OSCS made, then added to heparin? 

• Or was a heparin/chondroitin mixture oversulphated? 

Samples 

Code %OSCS 

02/09/012/0030‐D01 Netherlands 14.5 

02/09/012/0031‐D01 Netherlands 17.3 

02/09/046/0099‐D01 Ireland 29.4 

02/09/046/0100‐D01 Ireland 8.8 

Bulk heparin containing material of Chinese origin; 

OSCS content estimated d by b EDQM NMR method h d( (McEwen et al. l 

Pharmeuropa Bio 2008‐1). 

11/29/2010 

10

Methods 

Extracted contaminant from heparin by dropwise EtOH addition, 

which precipitated only the material with highest sulfate 

content 

Tested using a battery of physicochemical techniques, including 

NMR NMR, gel permeation chromatography chromatography, and anion exchange 

chromatography. 

1 H NMR spectroscopy of 0099‐D01 

at 500 MHz 

o 

Heparin 

- 

O 

O 

O 

H OD 

H 

O H 

H 

H 

S 

H 

- 

O 

O 

O 

OSCS 

- 

O O 

S 

H O 

O 

H 

DO H 

H 

ND2 H 

O O 

S 

- 

O 

O 

Na + 

Na + 

Na 

n 

+ 

O 

O 

S O 

H 

O 

O 

. 

H 

H 

COO 

O 

H 

O 

H O 

ND2 H 

H 

H 

O 

O C 

- 

H 

H 

CH3 O 

- 

- 

O 

O 

O S 

O 

. O O 

S 

O 

S 

O 

O O 

- 

Na + 

Na + 

n 

o 

11/29/2010 

11

Extracted OSCS from 0099‐D01 

1 H NMR at 500 MHz of OSCS extracted from 

contaminated heparin. 

The spectrum is in accord with published 

spectra of OSCS (Maruyama et al. 1997). 

Na + 

Na + 

Na + 

O 

O 

S O 

H 

O 

O 

. 

H 

H 

COO 

O 

H 

O 

H O 

ND2 H 

- 

- 

O 

O 

O S 

O 

2 

. O H 

O 

H O C H 

O 

- O 

S 

H 

H 

CH 

S 3 

O O O O 

n 

- 

Na + 

Na + 

• The OSCS in the contaminated heparin is a relatively 

homogeneous product, completely or almost completely 

sulphated. This is not easy to achieve deliberately, let alone by 

accident 

Molecular weights: 

GPC chromatograms 

High molecular weight 

component 

0099 D01 

5 th IS (pure heparin) 

OSCS from 0099 D01 

11/29/2010 

12

Conclusions (1) 

• Our evidence indicates that, for the samples of contaminated 

heparin that we analysed, OSCS was prepared separately from 

heparin then mixed with it. 

• The contamination did not arise from oversulphation of a 

heparin/chondroitin sulphate mixture. 

• OSCS contaminated heparin has higher activity in anticoagulant 

assays based on sheep plasma than in similar assays using 

human plasma p or purified p reagents g 

• OSCS has low activity in assays that use purified reagents. 

Conclusions (2) 

• Working independently, over a relatively short timespan, the 

USP and the EP developed similar strategies for monograph 

revision 

• Revised heparin monographs in both compendia include 

orthogonal physicochemical methods to protect against 

contamination with OSCS, and against gross contamination with 

other heavily sulphated non‐heparin polysaccharides 

• Higher minimum specific activity criteria for heparin heparin, adopted 

by both the USP and the EP, also protect against gross 

contamination, as does the use of a potency assay based on 

purified reagents 

11/29/2010 

13

Acknowledgements 

• Carolyn Swann 

• Elaine Gray NIBSC 

• John Hogwood 

• Nick Sutcliffe 

• Andy Charvill MHRA 

i S j k S 

• Anita Szajek USP 

• USP Heparin Advisory Panel Members 

11/29/2010 

14

DEG, EG and the Compendia’s 

Role in Control of Low-Level Toxic 

Species in Small Molecules 

Catherine Sheehan, M.S., 

Director, Excipients, USP 

USP Sponsored Workshop at AAPS-PWSC 

Hilton New Orleans 

November 13-14, 2010 

Outline 

• Glycerin adulteration results in the 1938 

Amendment to the FFD&C Act 

• FDA request to strengthen USP Glycerin 

Monograph 

• Introduction of an identification test to determine 

Limit of Diethylene glycol (DEG) and Ethylene 

glycol (EG) 

– “Hi “High h priority” i it ” Monographs M h identified id tifi d for f modernization d i ti 

– Summary of method development 

• USP Monograph modernization 

• Conclusions 

11/29/2010 

1

History of adulteration with Diethylene Glycol 

Country Year Incident 

USA 1937 “Elixir sulfanilamide” – 107 deaths 

Resulted in the implementation of the1938 Amendment to the FFD&C Act 

South Africa 1969 Sedative formulated with DEG – 7 deaths 

It Italy l 1985 DEG iin wines i ffrom AAustria t i – no kknown ddeaths th 

India 1986 Medicinal glycerin laced with DEG – 14 deaths 

Nigeria 1990 Acetaminophen syrup containing DEG – 40 deaths (some 

sources say 200 deaths) 

Bangladesh 1990-2 Acetaminophen syrup containing DEG – 339 deaths 

Haiti 1995/6 Cough medicine containing DEG – 85 deaths 

Panama 2006 Cough and anti-allergy syrup containing DEG – 46 deaths (116 

or o 365 according acco d g to ot other e 

USA 2006/7 Toothpaste containing DEG – no deaths 

Panama 2007 Toothpaste containing DEG – no deaths reported 

Nigeria 2008/9 Teething formula contaminated with DEG from propylene glycol 

– 84 deaths 

Bangladesh 2009 Paracetamol syrup to children adulterated with diethylene 

glycol. Twenty-four children reported dead 

The Problem: Even to the Trained Professional… 

Ethylene Glycol (“Antifreeze”) 

POISON! 

• Light colored 

• Slightly viscous liquid at room temp. 

• Sweet taste 

Glycerin (Glycerol) 

Edible and GRAS 




Albinus D’Sa, Ph.D., FDA, 2008 ASM Kansas 

Propylene Glycol 

Edible and GRAS 




Diethylene Glycol (“Antifreeze”) 

POISON! 




11/29/2010 

2

USP Response to Haiti incident for USP Glycerin Monograph 

• In the late 1990s, in response to the Haiti 

incident, USP revised the Glycerin monograph 

to include: 

– Identification section: Addition of 

“Identification Test B”. Glycerin Identification 

by retention time 

– Impurities section: Addition of the “Limit of 

DEG and Related Compounds Compounds” Test 

• A capillary gas-chromatographic (GC) method with 

flame ionization detection (FID) 

• NMT 0.1% DEG 

More adulteration 

• Panama 2006 

– Cough and anti-allergy syrup containing DEG – 46 deaths 

(116 or 365 according to other reports 

• USA 2006/7 

– Toothpaste containing DEG – no deaths 

• Panama 2007 

– Toothpaste containing DEG – no deaths reported 

• Nigeria 2008/9 

– Teething formula contaminated with DEG from propylene 

glycol – 84 deaths 

• Bangladesh 2009 

– Paracetamol syrup to children adulterated with diethylene 

glycol. 24 children reported dead 

11/29/2010 

3

FDA Request 

• April 2007: FDA request USP to modernize the Glycerin 

Identification section to include the identification and 

quantitation of Diethylene glycol (DEG) in glycerin. 

• JJan. 2009 2009: FDA letter l tt requested t d a modernization d i ti to t 

both Sorbitol Solution and Propylene Glycol consistent 

with the update to the USP Glycerin Monograph 

• Rationale: GMPs allow the use of Identification testing 

alone, by dosage form manufacturers, for raw 

material(s) qualification 

– manufacturers could therefore not deviate from the 

DEG limit since this would be an aspect of identity. 

• Challenge: Complex issue relating to ‘requirement’ that 

contaminant/adulterant be considered part of an article’s 

Identification 

FDA’s May 2007 Guidance Regarding DEG Contamination of Glycerin 

Reiterates §211.84(d)(2) requirement 

for specific ID testing when not 

performing full USP testing 

Recommends intimate knowledge g of 

the supply chain 

Testing has to be capable of detecting 

DEG 

Applies to all recipients of Glycerin 

USP, not only those who formulate or 

compound 

Traceability 

8 

11/29/2010 

4

21 CFR Part 211 - CGMP Requirements for Drug Manufacturers 

(Subpart E) Control of Components… 

� 21 C.F.R. § 211.84(d) 

– “At least one test shall be conducted to verify the identity of each 

component of a drug product. Specific identity tests, if they exist, 

shall be used.” 

• requires that manufacturers of drug products detect and 

quantify any DEG present both at the time of manufacture and 

upon receipt at the point of transfer to another party. 

• manufacturers cannot deviate from the DEG limit since this is 

an aspect of identity identity. Cannot label away from identity! 

• In contrast, if DEG detection and quantification is solely part of a 

purity (impurity) test, a manufacturer need not include as part of 

its identity testing 

USP-NF Articles: FDA Identified ‘High-Priority’ for Adulteration with DEG and EG 

Maltitol Solution (1) (H) 

Sorbitol Solution (1) (H) 

Sorbitol sorbitan solution (1) (H) 

Noncrystallizing sorbitol solution (1) (H) 

Propylene glycol (2) (H) 

Propylene glycol dilaurate (4) (M) 

Polyethylene glycol (3) (M) 

Lactitol (1) (L) 

Maltitol (1) (L) 

SSorbitol bit l (1) (L) 

Polyethylene glycol monomethyl ether (4) (L) 

Diethylene glycol monoethyl ether (4) (L) 

Diethylene glycol stearates (4) (L) 

1- Sugar alcohols 

2- Propane diols and triols 

3- Polyols (polyethylene glycol) 

4- Derivatives of categories 1-3 

The risk levels for undetectable 

contamination are categorized as 

H – high 

M – medium 

L – low 

11/29/2010 

5

Comments from CDER Office of Compliance on the USP List 

• Highest risk ingredients 

– aqueous solutions or liquids that can be readily 

spiked with DEG or EG 

– similar physical properties in terms of viscosity 

– may be sweet tasting and thus make their way 

into solutions, syrups and elixirs at relatively 

high levels of use 

• IIG search for ingredients deemed high high-risk, risk 

revealed that they are in fact used at very large 

amounts such that toxic levels are readily 

achieved. 

Ethylene Glycol impurity 

• While FDA’s original request to USP did not include 

controlling EG in the Identification test, EG was 

added to the list of the “high-priority” high priority excipients 

• Due to its potential presence as a byproduct of 

the high pressure catalytic hydrogenation of 

hydrolyzed corn syrups used to manufacture 

sugar alcohols 

• High toxicity 

• Similar physical and chemical properties to DEG. 

11/29/2010 

6

Monograph modernization of “High-Priority’ monographs 

A total of seven excipient monographs (includes 1 new 

monograph) were categorized as ‘high-priority’ for modernization 

of Identification test: 

– Glycerin 

– Sorbitol Solution 

– Sorbitol Sorbitan Solution 

– Noncrystallizing Sorbitol Solution 

– Maltitol Solution 

– Propylene Glycol 

– Hydrogenated Starch Hydrolysate (new monograph in PF) 

Placement of Limit for DEG/EG in the Identification section 

would help control these Low-Level Toxic impurities 

Summary of validated method development 

� Two capillary gas chromatographic methods with FID 

were developed and validated for analyzing EG and DEG. 

– GGlycerin and Propylene glycol: SSample 

preparation easy: 

dissolve in methanol and direct inject into GC. 

– Polyols: Sorbitol solution, Sorbitol sorbitan solution, 

Noncrystallizing sorbitol solution, Maltitol solution and 

Hydrogenated Starch Hydrolysate 

• Sample preparation more difficult. Two steps: extraction of EG and 

DEG and d precipitation i it ti of f the th polyol l l using i an acetone-water t t (96:4, (96 4 

v/v) mixture as solvent. 

� Limit tests were validated for specificity, accuracy, method 

precision, and LOD. 

11/29/2010 

7

REVISION BULLETTIN 

IPR 

Monograph modernization via Revision Bulletin and IPR 

• Glycerin 

– Official date May 1, 2009 

• Sorbitol Solution 

• Sorbitol sorbitan solution 

• Noncrystallizing sorbitol solution 

• Propylene glycol 

– Official date February 1, 2010 (same DEG and EG 

limits) 

• Maltitol Solution 

– Official date August 1, 2010 (same DEG and EG limits) 

• Hydrogenated Starch Hydrolysate 

– PF 37(1) [Jan. – Feb. 2011] (same DEG and EG limits) 

USP Monograph modernization efforts 

• Many excipient monographs include non-specific 

assay and/or identification tests, some do not have 

identification tests 

• Need to re-evaluate antiquated methodologies for 

excipients with a view to modernization. 

• FDA strongly believes that monographs utilizing 

outdated analytical procedures are vulnerable to 

economically motivated adulteration (EMA) 

• Current advancements in science and technology 

can help to control low level toxic impurities such as 

DEG and EG 

11/29/2010 

8

USP Monograph modernization efforts 

• Need to re-evaluate antiquated methodologies for 

excipients with a view to modernization 

• Need to develop and introduce specific and 

selective method(s) for Assay and/or Identification 

tests where missing or deficient in a monograph 

• Modernization of tests can help to accomplish this 

task 

• This approach helps determine if the material 

contains t i impurities i iti or contaminants t i t such h as EG 

and DEG 

Conclusions 

• Strengthening and conformance to compendial 

specifications is one of the tools necessary to 

help prevent the next economically motivated 

adulteration with low level toxic species that is 

part of USP’s efforts to ensure safe medicines 

�Need help from stakeholders like you! 

11/29/2010 

9

11/29/2010 

10

Rx-360 

An International Pharmaceutical 

Supply Chain Consortium 

Working Together for Patients 

USP Sponsored Workshop 

AAPS AAPS–PWSC PWSC 

November 13, 2010 

New Orleans, LA 

Patient safety must never 1 be compromised as a 

competitive advantage 

Illicit Activity is Present and 

Threatens the Safety of Medicines 

$1000 

Counterfeit 

DVDs 

$10,000 

• The criminal element is present, 

active active, and business savvy 

• ‘Benign’ consumer actions may 

have tragic consequences 

Illegal Counterfeit 

drugs medicine 

$100,000 $1,000,000 

2 

1

Tragic Consequences 

Not If… But When and Where 

May 7, 2007 

80 children die in Haiti due to contaminated 

glycerin in acetaminophen syrup 

4 

2

Rx-360 Mission 

Create and monitor a global quality 

system that meets the expectations of 

industry and regulators, that assures 

patient safety by enhancing product 

quality and authenticity throughout the 

ssupply ppl chain 

Rx-360 Operating Model 

• Designed to meet competition law requirements 

• Not intended to replace regulatory systems or 

oversight 

• International not-for-profit organization 

• Volunteer based 

• Broad and inclusive membership 

– Small and large companies 

– Branded and generic 

– Suppliers, distributors, brokers and manufacturers 

• Companies are members not individuals 

5 

6 

3

• Collaborative 

Rx-360 Key Principles 

• Rx-360 does not want to compete but to leverage 

strengths of all parties 

• Flexibility 

• Members can participate as they desire 

• Open Architecture and Transparent 

• IP will be shared freely with the public 

Rx-360 is Having an Impact 

• Detecting Detecting, Deterring and Disrupting 

while improving quality 

1. Information Sharing 

2. Technology Sharing 

3. Standard Setting 

4. Audit Sharing 

7 

8 

4

Since April 2009 Rx-360 Has Distributed 

Information to Registered Users 

9 

Rx-360.org Shares Information That Can 

Benefit Us All: Swine Flu Impact Analysis 

10 

5

Rx-360.org Shares Technology That Can 

Benefit Us All: Acetonitrile Method 

Determination of Adulteration of Acetonitrile using Gas Chromatography with Thermal Conductivity Detection 

11 

Standards Are Required to 

Create a Level Playing Field 

• Id Identify if existing i i bbest practices i 

• Endorse best practices 

• Implement quickly 

• Continuously refine after successful 

iimplementation l t ti 

“Perfection is the enemy of good.” 

12 

6

Sharing Supplier Audits Will Increase 

Compliance, Quality and Efficiency 

1. Sharing existing audit information from one 

member b with ith many members b 

2. Sponsor audits are initiated by a single 

pharmaceutical firm 

3. Audits led by Rx-360 are initiated by the 

consortium based on input from all members 

44. Subscription audits are existing audits that are 

redacted and placed into a secure database for 

member access 

Numerous 1 or 2 day audits 13will 

be replaced with fewer & 

more thorough audits thus eliminating “Audit Fatigue” 

We Need to Secure the Supply 

Chain from Many Things 

• Accidental adulteration 

• Unethical participants 

• Economically motivated adulteration 

• Sabotage and terrorism 

14 

7

To secure the supply chain 

11. First use common sense 

2. Then embrace new ideas 

3. Adopt advanced technologies; and 

4. Always collaborate 

15 

Common sense dictates that you 

understand your supply chain 

• Who is the manufacturer? 

• Where is it manufactured? 

• How many links are in your supply 

chain? 

• Is your supplier transparent? 

• How is the material transported and 

stored? 

Every step in the supply chain 16 represents a potential risk 

8


do business with a trusted partner 

• Due diligence 

• Build and maintain a relationship 

• Routine contact 

• Provide routine oversight 

When a trusted partner is not available… 

17 


have a robust risk management process 

• Apply resources based on risk 

• When there is an unacceptable risk 

- Move them up 

- Move them out 

- Install risk mitigation 

When there are doubts act quickly 

18 

9

Common sense dictates the 

use of tamper evident seals 

• Tamper evident seals 

– Not a K-mart zip tie 

– Not Ace Hardware duct tape 

– Not a piece of string 

– Not a rubber band 

– Not a knotted plastic bag 

• SSeals l should h ld hhave 

a 

unique number 

Most importantly seals should be verified 

19 

for each shipment and every container 

There are many new ideas that can be 

embraced to secure raw materials! 

• Right Size Testing 

– Need to change the 

paradigm to drive more 

robust screening 

• Skip lot, abbreviated, 

reduced testing, etc. 

– Compendia testing may 

not be sufficient ff 

– What about NIR, 

Raman, Mass Spec 

GC, NMR, etc. 

20 

10


embraced to secure raw materials! 

• Photographic 

Lib Libraries i 

Increase a sense of awareness and increase 

21 

the number of eyes looking for problems 


embraced to secure the supply chain! 

• Pedigree 

• Efpia Pilot 

– 4 months 

– 14 companies 

– 25 pharmacies 

– 95,000 packs scanned 

• Results 

– http://www.efpia.eu 

– MModel d l worked k d 

– Identified fake and expired packs 

– Did no increase pharmacy’s workload 

– High user acceptance 

22 

What can we do to apply pedigree 

to raw materials? 

11


embraced to prevent cargo theft! 

• Two drivers 

• Chase cars 

• Can’t stop for at least 4 hours or 200 miles 

• Inform the driver they are being tracked 

• Use covert tacking devices and services 

Harden your transportation so criminals 

23 

go to easier targets 


embraced to prevent cargo theft! 

• Janssen-Cilag has developed 

a new web-based web based tool for 

customs to check whether a 

shipment is fake or genuine. 

www.dvp-eu.com 

• Collaboration with customs officials and was roadtested 

last year by 17 customs officials in 10 EU 

countries 

• Janssen-Cilag will sometimes change product 

packaging every year, as this is in itself a protection 

against counterfeiting, but this makes it even more 

hard to ensure that all 27 EU customs authorities have 

up-to-date information. 

24 

Janssen-Cilag has been trying to encourage 

other drug makers to join in with the project 

12

New technologies are being implemented 

FedEx SenseAware provides real time monitoring 

• Sensing: In flight GPS real time 

communications, and sensors for 

temperature, humidity, light, barometer, 

and accelerometer 

• New-to-world information service 

and interface 

• SA Alliance Certification: Programs 

and processes to certify 3rd party’s 

hardware, software, and accessories, 

along with management of IP-licensing 

• Multiple SA Solutions: Integrated and 

configurable sensors and accessories 

available for purchase, such as 

packaging, pallet, and container 

systems that address different 

environmental, energy, security, and 

communication management scenarios25 

New technologies are being implemented 

FreightWatch offers product tracking 

• Tracks shipments vs a pre-defined 

route called a geofence g 

• Provides alarms if the geofence is 

violated 

• Emergency Response Center are 

on-call 24x7 to coordinate 

recovery of stolen cargo with a 

nationwide network of law 

enforcement agencies g and cargo g 

theft investigators 

26 

13

To protect the patients you serve, the 

product you make and your company 

I strongly encourage you 

– Apply common sense 

– Embrace new ideas 

– Evaluate new technologies 

– Start collaborating by convincing 

you company to join Rx-360! 

Patient safety should never be a competitive 

27 

advantage and can never be achieved by…. 

Over 40 Organizations Have 

Signed On Thus Far 

Members Observers 

Manufacturers (22) Suppliers (15) 

Auditors (6) 

• Abbott 

• Archimica Group 

• PSC Biotech Corp. Corp 

• Alcon 

• BASF 

• Regulatory Compliance Associates 

• Amgen 

• Fagron 

• RMC Pharmaceutical Solutions Inc. 

• Amylin 

• FreightWatch 

• Safis Solutions LLC 

• AstraZeneca 

• GE Healthcare 

• SQA Services Inc. 

• Baxter 

• Hovione 

• The Weaver Group, Inc. 

• Bayer 

• Labochim 

• 

• 

• 

• 

• 

• 

• 

• 

• 

• 

• 

BiogenIdec 

Boehringer Ingelheim 

BMS 

Cephalon 

Eli Lilly 

GSK 

Hospira 

Johnson & Johnson 

Merck & Co. 

Novartis 

OSO Biopharmaceuticals 

• 

• 

• 

• 

• 

• 

• 

• 

• 

LifeConEx 

Mallinckrodt Baker 

Merck KGaA 

Reliable 

Biopharmaceutical Corp. 

Sigma Aldrich 

SSpectrum Ch Chemical i l 

TempTime 

VWR 

West 

Associations (8) 

• APIC 

• Council for Responsible Nutrition 

• European Fine Chemicals Group (EFCG) 

• European Generic Medicines Association (EGA) 

• IPEC Americas 

• IPEC Europe 

• NSD Bio 

• Parenteral Drug Association (PDA) 

• Pharmaceutical Quality Group (PQG) 

• Pfizer 

• Sanofi-Aventis 

• Takeda 

• Watson 

28 

Rx-360 is now over 50 Organizations Strong 

14

We Must Act Now 

� There is a significant un-met need 

� Minimal costs are required to start-up and operate 

� The consortium allows us to collaborate to serve 

patients 

� The consortium has generated enthusiasm and 

excitement it t 

29 

Register as a User on www.Rx-360.org 

and Receive Valuable Industry News 

30 

15

Thank you 

For More Information 

31 

Jim Jamieson 

Drinker, Biddle & Reath 

jim.jamieson@dbr.com 

+1 (202) 230-5189 

16

Recent Health Canada Activities 

Related to Misidentification of Black Cohosh 

Pauline Lacroix, 

A/Manager HBFB Inspectorate, Health Canada 

Presented to: USP Sponsored Workshop at AAPS-PSWC Meeting 

November 13, 2010 

Outline 

• Introduction 

• Adverse Reactions 

• Reports of Mis-identification 

• Analytical Methodology/Results 

• Regulatory Actions 

• Conclusion 

2 

1

Introduction 

Figure 1. Black Cohosh 

• Black Cohosh (Actaea ( 

racemosa L., synonym: 

Cimicifuga racemosa (L.) Nutt.) 

is a North American plant 

whose root has a long tradition 

of medicinal use. 

• Sold widely y to relieve 

symptoms of menopause 

International Adverse Reaction Reports 

• In recent years years, over 80 international cases of 

hepatotoxicity (including liver transplants), suspected 

as being associated with products labelled as 

containing Black Cohosh, have prompted reviews of 

this herb’s safety. 

3 

4 

2

Review of Canadian Adverse Reactions 

• Four serious Canadian cases of hepatotoxicity p y associated with 

the use of Black Cohosh were reported to Health Canada as of 

June 2007. 

• The causality was assessed as “possible” in three cases (due to 

confounding concomitant medications and other medical 

conditions) and as “probable” in one case, according to WHO 

causality assessment criteria. 

• The probable p case of hepatitis p had no identifiable confounders, 

and was associated with a time to onset of 9 days following daily 

use of a product labelled as containing 100 mg Black Cohosh 

root extract/powder. 

Reports of Mis-labeled Products 

• It had been shown recently that some commercial 

products do not contain authentic Black Cohosh, but 

rather other species of Actaea (e.g. Jiang et al., 

2006). 

• This raises the question as to whether or not certain 

adverse reactions are related to authentic Black 

Cohosh. 

5 

6 

3

An Opportunity 

• To our knowledge, knowledge no regulatory agency had linked a 

reported adverse reaction to the testing of the 

product involved 

• Was this specific probable case associated with 

authentic Black Cohosh, or not? 

Coordination of Efforts within HC 

Marketed Health Products Directorate (MHPD) 

Natural Health Products Directorate (NHPD) 

Inspectorate 

- Regions - Longueuil Laboratory 

- Ontario Region 

- Western Region 

- Ottawa - Laboratory Programme Coordinator 

- Drug Compliance Verification and 

Investigations Unit 

7 

8 

4

Product: Black Cohosh 

Samples of Same Lot Associated with Case 

Medicinal Ingredients (from label): 

Each capsule contains: 

Standardized Black Cohosh Root Extract (2.5% Triterpenoids) 

(Actaea racemosa L.) 20 mg 

Black Cohosh Root Powder (Actaea ( racemosa L.) ) 80 mgg 

Samples of Same Lot Associated with Case 

Sample #2567-00860 

- retained sample from manufacturer 

- sealed white plastic bottle (180 capsules) 

Sample #2568-00861 

- purchased at retail retail, Scarborough 

- sealed white plastic bottle (60 capsules) 

9 

10 

5

Selection of Marker Compounds 

Source: Jiang et al. (2006), J. Agric. Food Chem., 54:3242-3253 

Method 

Method: Black Cohosh, Content of triterpene glycosides 

Pharmacopeial Forum 32(4) 1130-1132 

(modified: did not use an ELS detector) 

Equipment: LC Waters Alliance 2690 

Photodiode array detector 996: 205 nm 

MS: Waters Integrity system 

Thermabean detector 

Source temperature 275.0°C 

Nebulizer set point 75.0 °C 

Expansion Region Setpoint 80 80.0 0 °CC 

Gain 100.0 

Sampling rate 2.0 

Mass 55 – 500 m/z 

Column: Waters, YMC ODS-AQ, 5µm, 120Å, 4.6X250 mm 

11 

12 

6

Mobile phase: 

Method (cont’d) 

A : WWater t 

B : 0.05% Trifluoroacetic acid 

C : Acetonitrile 

Injection 20µl 

Flow 1.4ml/min on column 

Flow Splitter 0.4ml/min on PDA and MS. 

Gradient table: 

Time Flow %A %B %C 

0.00 1.40 0.0 80.0 20.0 

8.00 1.40 0.0 80.0 20.0 

8.10 1.40 68.0 0.0 32.0 

15.00 1.40 68.0 0.0 32.0 

55.00 1.40 36.0 0.0 64.0 

65.00 1.40 5.0 0.0 95.0 

70.00 1.40 5.0 0.0 95.0 

85.00 1.40 0.0 80.0 20.0 

100.00 1.40 0.0 80.0 20.0 

Method – Ions Monitored 

Marker Retention 

Time (Min.) 

Ions Monitored (m/z) ( ) 

Cimifugin 8.73 306.0 247.0 229.0 

Actein 32.69 483.0 466.0 423.0 

23 23-epi-26-Deoxyactein i 26 D t i 33 33.04 04 467 467.0 0 450 450.0 0 437 437.0 0 

Cimiracemoside C 36.72 470.0 427.0 383.0 

13 

14 

7

Region of Cimifugin 

(not detected) 

Figure 2. Full scan spectrum of USP Black Cohosh Standard 

Locus oof 

Cimerocemide 

C 

Figure 3. Full scan spectrum of Black Cohosh Sample 

Actein 

23-epi-26-ddeoxyactein 

Cimirocemmide 

C 

15 

16 

8

17 

18 

9

Results 

Marker Compound Actaea Other USP Samples 

racemosa L. Actaea Powdered 2567-00860 

( (Black species i Black 22568-00861 68 00861 

Cohosh) 

Cohosh 

Extract RS 

Actein + In some + + 

23-Epi-26-deoxyactein 

(a.k.a. 27-deoxyactein) 

+ In some + + 

Cimifugin g 

- In some Asian - + 

species 

Cimiracemoside C + - + - 

Results 

• The samples were also screened for undeclared 

pharmaceutical ingredients, adulterants and 

contaminants using routine screening methods but 

none were identified by the laboratory. 

19 

20 

10

Discussion 

Important considerations for identification methods: 

• Validated for specificity – they should be able to distinguish 

Black Cohosh from other Actaea species and able to detect 

indications of other Actaea species in the presence of Black 

Cohosh 

• Use of well characterized reference materials - 

– e.g. USP Powdered Black Cohosh Extract 

– For chemical methods, Cimiracemoside C and Cimifugin 

hhouse standards t d d should h ld bbe verified ifi d ffor id identity tit ( (e.g. mass 

spectrum, UV spectrum, NMR) 

• Specify appropriate control samples and blanks during 

analyses 

Discussion (Cont’d) 

Important considerations for LC-MS LC MS methods 

• Must be able to resolve the compounds of interest, 

especially Cimiracemoside C and Cimifugin from other 

compounds in Black Cohosh 

• Selection of appropriate ions: The various species of Actaea 

contain many structurally similar compounds (hence the 

possibility of having common daughter ions) and isomers of 

the compounds of interest (possibility of compounds with the 

same molecular weight) 

• Monitoring several ions, including the parent ion and key 

daughter ions is recommended 

21 

22 

11

Discussion (Cont’d) 

Important considerations for LC-MS LC MS analyses of 

products: 

- Ensure the reference materials are run on the same day 

under the same conditions as the samples 

• Spiking experiments for confirmation of presence or absence 

of the compounds of interest 

Conclusions from the Laboratory Analysis 

The samples investigated do not contain Black Cohosh Cohosh. 

They may contain some other Asian Actaea species. 

23 

24 

12


Recommendation #1 

• Communicate the analytical findings to the manufacturer, and 

request information on present/future quality control procedures 

to ensure future products contain only authentic Black Cohosh. 


• Require specific species identification as part of Black Cohosh 

Product License Applicants submitted to NHPD NHPD, and determine 

the need for ensuring the quality of Black Cohosh products 

currently available on the Canadian market. 

Actions by the Regulator in Response to 


• The manufacturer was sent a Natural Health 

Products Regulations Section 16 (post-licensing 

safety issue) Notice Notice by NHPD which 

summarized the possible risks to health presented by 

the product, the results of the HPFB Inspectorate 

laboratory testing, and the necessary remedial 

actions to maintain their marketing authorization. 

25 

26 

13


Recommendation #1 (Cont’d) 

• The manufacturer was informed that the method they 

previously used to identify the medicinal ingredient is 

no longer considered adequate to accurately identify 

Actaea racemosa. 

• The manufacturer was asked to confirm the method 

that will be used to correctly identify the medicinal 

ingredient Actaea racemosa in Black Cohosh 

products. 



• NHPD recognized that there are numerous unambiguous 

methods th d which hi h can bbe used d tto id identify tif Bl Black k CCohosh h h and d 

provided references below to some such methods: 

• He K, Zhen B, Kim CH, Rogers L, and Zheng Q 2000. Direct analysis and 

identification of triterpene glycosides by LC/MS in Black Cohosh, Cimicifuga 

racemeosa, and in several commercially available Black Cohosh products. 

Planta Med 66(7): 635-640. 

• He K, Pauli GF, Zheng B, Wang H, Bai N, Peng T, Roller M, and Zheng Q. 2006. 

Cimicifuga species identification by high performance liquid chromatographyphotodiode 

array/mass spectrometric/evaporative light scattering detection for 

quality control of Black Cohosh products. J Chromatogr A 1112(1-2):241-254. 

• Jiang B, Kronenberg F, Nuntanakorn P, Qiu M-H, and Kennelly EJ. 2006. 

Evaluation of the botanical authenticity yand pphytochemical y pprofile 

of Black 

Cohosh products by high-performance liquid chromatography with selected ion 

monitoring liquid chromatography-mass spectrometry. J Agric Food Chem 

54:3242-3253. 

• Avula B, Ali Z, Khan IA. 2007. Chemical Fingerprinting of Actaea racemosa (Black 

Cohosh) and Its Comparison Study with Closely Related Actaea Species A. 

pachypoda, A. podocarpa, A. rubra) by HPLC. Chromatographia 66(9/10):757-762. 

27 

28 

14



• The manufacturer was asked to confirm when a new method 

would ld bbe iincorporated t dffor all ll ffuture t bbatches t h of f products d t th that t 

contain the medicinal ingredient Black Cohosh. 

• NHPD also requested that details of the validated methodology 

and the test results for at least 3 batches of each product 

containing Black Cohosh be provided using the revised 

methodology. 

• Given the findings of the Health Products and Food Branch 

Inspectorate’s analysis of the product associated with the 

serious adverse reaction, it was requested q that the 

manufacturer provide information to confirm the safety of other 

lots of the same product and of their other products on the 

Canadian market which contain the medicinal ingredient Black 

Cohosh. 


Recommendation #1 - update 

• After an initial contestation of the Inspectorate 

laboratory’s results by the manufacturer’s contract 

laboratory and a review of their validation data by the 

Inspectorate, the testing laboratory contracted by the 

manufacturer agreed with the Inspectorate that the lot 

of interest did not contain Black Cohosh and has 

since developed and validated a new method. 

• Compliance verification is ongoing. 

29 

30 

15



� Section 16 letters were sent to other licensees requesting 

further evidence of identity testing and adverse reaction 

reporting 

� A standard line has been developed to use in Information 

Request Notices for all Product Licence Applicants with 

Black Cohosh, based on the content of the Section 16 

letters, requiring unambiguous identification of Black 

Cohosh as Actaea racemosa L. by one of the selected 

HPLC methods or another equally unambiguous method 

such as DNA fingerprinting 



• A revision to the NHPD Black Cohosh Monograph was published online 

(htt (http://webprod.hc-sc.gc.ca/nhpid-bdipsn/monoReq.do?id=44&lang=eng) 

// b d h / h id bdi / R d ?id 44&l ) 

requiring the identity of Black Cohosh to be determined unambiguously as 

Actaea racemosa L. and not any other species, at either the raw 

material stage or the finished product stage, in accordance with one of 

the following methods: 

� He K, Zhen B, Kim CH, Rogers L, and Zheng Q 2000. Direct analysis and identification of 

triterpene glycosides by LC/MS in Black Cohosh, Cimicifuga racemeosa, and in several 

commercially available Black Cohosh products. Planta Med 66(7): 635-640. 

� He K, Pauli GF, Zheng B, Wang H, Bai N, Peng T, Roller M, and Zheng Q. 2006. 

Cimicifuga species identification by high performance liquid chromatography-photodiode 

array/mass spectrometric/evaporative light scattering detection for quality control of Black 

Cohosh products. J Chromatogr A 1112(1-2):241-254. 

� Jiang g B, , Kronenberg g F, , Nuntanakorn P, , Qiu Q M-H, , and Kennelly yEJ. 

2006. Evaluation of the 

botanical authenticity and phytochemical profile of Black Cohosh products by highperformance 

liquid chromatography with selected ion monitoring liquid chromatographymass 

spectrometry. J Agric Food Chem 54:3242-3253 

� Avula B, Ali Z, Khan IA. 2007. chemical fingerprinting of Actaea racemesa (Black Cohosh) 

and Its comparison study with closely related Actaea species. ( A. pachydpoda, A. 

podocarpa, A. rubra) by HPLC. Chromatographia 66 (9/10) : 757-762 

31 

32 

16


Recommendation #2 - Update 

• Health Canada provided an update on ADRs in the 

January 2010 edition of the Canadian Adverse 

Reaction Newsletter. (http://www.hc-sc.gc.ca/dhpmps/medeff/bulletin/carn-bcei_v20n1-eng.php) 

Conclusion 

• Mis-identification of Black Cohosh, which may also involve 

deliberate substitution of less expensive p Asian species p for the 

authentic North American Actaea racemosa, has been 

conclusively demonstrated in products marketed in the U.S. and 

in Canada. 

• This may be one factor in the adverse reactions, along with use 

of the wrong plant part (aerial parts rather than roots), 

contamination, adulteration, and idiosyncratic reactions of 

consumers. However, inherent toxicity, while unlikely, cannot be 

ruled out at this time 

• This is the first case Health Canada is aware of where an 

adverse reaction report regarding a specific product labelled as 

Black Cohosh has been followed up with laboratory 

determination that the species involved was not authentic Black 

Cohosh 

33 

34 

17

Conclusion 

• By working together to bring this issue to a 

successful f l conclusion, l i th the MMarketed k t d HHealth lth PProducts d t 

Directorate, Natural Health Products Directorate, 

Health Products and Food Branch Inspectorate and 

the Regions and Programs Branch have 

demonstrated the benefits of the Natural Health 

Products Program approach to regulation. 

• Thi This iis an excellent ll t example l of f cooperation ti bbetween t 

Health Canada and the USP to ensure the quality of 

natural health products. 

NHPD: Robin Marles 

Semir Omar 

MHPD: Mano Murty 

Scott Jordan 

Shahid Perwaiz 

Danika Painter 

Semir Omar 

Special thanks to: 

Inspectorate: Richard Bertrand 

for assistance in preparing this presentation 

35 

36 

18

Thank you! 

Pauline.lacroix@hc-sc.gc.ca 

37 

19

QUALITY CONTROL ON SAFETY AND 

EFFICACY OF CHINESE MATERIA MEDICINES 

Shen Ji 

Shanghai Institute for Food and Drug Control 

Address: 1500 Zhang Heng Road, Shanghai 201203, China 

Email: jishen2008@gmail.com 

Characteristics of Quality Control on Chinese 

Materia Medicine (CMM) 

I. Focus on the safety of TCM quality control. 

II. Strengthen the material basis of TCM and 

Achieve multi-component medicine, multi-target 

quality control. 

III. Improve the quality control of toxic and effective 

medicine. 

1 

2 

1

Ⅰ Focus on the safety of TCM quality 

control. 

Study on the standards of 

� heavy metals 

� biotoxins 

�� pesticide residues 

� organic residues of macroporous absorbent 

resin 

Study on analytical standards of heavy metals 

The standards in China and abroad 

2005 Ch Ch.P P 2010 Ch Ch.P P USP and BP 

Measured Methods AAS, ICP-MS AAS, ICP-MS, ICP-OES 

AAS, Colorimetry, 

ICP-MS 

Elements Pb, As, Hg, Cd, Cu Pb, As, Hg, Cd, Cu 1~6 

Monitoring species 

6 species, such as 

Glycyrrhizae Radix 

� Enrich and improve the 

detection method of heavy 

metals and harmful elements in 

CMM 

� Build a sound system of 

elements determination 

10 species, such as 

Ginseng Radix 

Limit 

All plants 

Element Pb Cd Hg As Cu 

(mg/kg)

Study on analytical standards of Aflatoxins 

2010 Ch.P 

� Improve the 

national standards 

�� Accordant to the 

level of 

international 

pharmaceutical 

standards 

2005 Ch.P 

2010 Ch.P 

South 

Korean 

Hong Kong 


Method 

HPLC- 

FLD 

HPLC- 

FLD 

Monitoring 

species 

Limit 

Chinese herbal 

medicines 

None None 

5 species 

B1≤5μg/kg, 

total ≤10μg/kg 

None 9 species B1≤10μg/kg 

HPLC- 

FLD 

8 species 

B1≤5μg/kg, 


USP TLC None None 

EP 

further study items 

HPLC- 

FLD 

All plants 

B1≤2μg/kg, 


Future focuses on the biotoxins 

� Determination of aflatoxin 

by PCD 

� LC-MS method of aflatoxin 

� Expand the variety of aflatoxin 

detected 

� Determination of ochratoxin 

avomitoxin, zearalenone, 

patulin 

Objectives 

Chinese 

Patent 

Medicine 

None 

�Enrich the detection 

methods 

�Eliminate the matrix 

interference, avoid 

false-positive 

�Help to ensure 

accurate and reliable 

results 

5 

6 

3

Control methods on pesticide residues 


2005 Ch.P 

The projected additional 

method in 2010 Ch.P 

USP, EP, JP, HK 

Pretreatment Ultrasonication, Homogenate extraction, 

Homogenate 

technology SPE ASE, GPC, Tandem SPE extraction, GPC, SPE 

Method GC 

Study 

species 

The number of o 

pesticides 

Comparison of the number of pesticides analyzed in domestic 

and international standards 

150 

100 

50 

0 

2 species 

24 

47 

GC (dual-ECD, dual-FPD, 

NPD), GC/MS, LC/MS/MS, LC 

105 

Studied More than 20 

species 

8 

1 

Standards 

34 

127 

GC, GC/MS 

All plants 

2005CP 

USP 

EP 

JP 

HKGL 

Our Project 

We analyzed 127 pesticides. Due to some other reasons, pesticide residues have not received 

Pharmacopoeia 2010 edition , we still continue to study faster, more exclusive method. 

Our goal is: improve detection methods of pesticide in Chinese medicine to the level of 

food pesiticide detection. 

MS and chromatogram profiles of pesticides 

Total ion chromatogram of a standard w 

ith 53 pesticides in GC-MS 

The chromatogram of a standard with 1 

5 organchlorines in GC-ECD 

Total ion chromatogram of a standard 

with 74 pesticides in LC-MS/MS 

The chromatogram of a standard with 19 o 

rganophosphorus in GC-FPD 

4

Analysis of organic residues of macroporous absorbent 

resin 

�Macroporous 

resin is often used 

iin the th production d ti 

process of extract 

�Organic residues of 

macroporous 

absorbent resin may be 

to cause a certain 

security risk to extract 

�Applied to the safety quality control , such as 

Notoginseng Radix et Rhizoma extract 

� Provide technical support for improving the quality 

standards of CMM extract 

�Establish a general 

analysis method for 

organic residues by 

headspace GC 

The GC profile of macroporous absorbent resin organic residues 

1. n-hexane 2. benzene 

3. tolune 4. p-xylene 

5. o-xylene 6. styrene 

7. 1,2-diethylbenzene 

8. divinylbenzene 

Ⅱ Strengthen the material basis of TCM and Achieve 

multi-component medicine, multi-target quality control. 

Use a few examples to illustrate these issues: 

� Substantial basis research achieved a comprehensive quality 

control of CMM 

� Chiral Mobile Phase Additives resolved the separation of the 

isomers of total glucosides in Centellae Herba 

� Control of related substances improved the quality standard of 

Menthol 

� Fingerprint 

Liquidum 

ensured the safe of Extractum belladonnae 

The quality control of traditional Chinese medicine have gone 

through three stages: No ingredients control, single index 

component control, and now the comprehensive multi-component 

control. 

5

Strengthen the material basis of TCM 

Tanreqing Injection—— Research method 

Identify the components in the total 

solid of the injection 

mainly composed of four extracts 

Extractum Scutellariae Siccus Extractum 

Lonicerae Siccus 

Flos Extractum Forsythiae Siccus 

Elucidate the substantial 

basis of the extracts 

Use the example of 

traditional Chinese 

medicine injection process 

to clarify how we conduct 

quality control. 

Clarify category composition and 

monomer of the injection 

Establish specific analytical methods 

Methodology 

Injection Extractum Herbal medicine 

Ensure safety, stability, effectiveness of CMM injections 

Extractum Bear 

Bile Powder 

Extractum Scutellariae Siccus- 8 compounds identified 

1 

2 4 

1 5,6,4’-OH;7-O-Glucuronic acid 

2 5,6-OH;7-O-Glucuronic , ; 

acid 

3 5-OH;6-OCH3 ;7-O-Glucuronic 

acid 

4 5-OH;7-O-Glucuronic acid 

5 5-OH;8-OCH3;7-O-Glucuronic 

acid 

6 5,6,7-OH 

7 5,7-OH;8-OCH3 

8 5,7-OH;6-OCH3 

3 

5 

6 

7 

8 

7 

6 

8 

� about 98% ingredients of the 

extractum were elucidated 

5 

O 

O 

3 

2' 

11 

the structure of their nucleus 

� Basically clarify the substantial 

basis of Extractum Scutellariae 

Siccus 

3' 

6' 

12 

4' 

5' 

6

Extractum Flos Lonicerae Siccus 

1 23 4 5 6 7 

1. neochlorogenic acid 

2. chlorogenic acid 

33. cryptochlorogenic acid 

4. not identified 

5. caffeic acid 

8910 11 12 

Total Ion Chromatography 

6. swerodie 

9. 3,4- Dicaffeoylquinic acid 

7. secoxyloganin 10. 3,5- Dicaffeoylquinic acid 

88. luteoloside 11 11. 44,5- 5- Dicaffeoylquinic acid 

12. Ethyl Caffeate 

12 Organic acid ingredients occupy 28%-39% 

1. forsythoside E 

2. forsythoside y D 

3. caffeic acid 

4. Group of peaks(forsythoside C and its isomers) 

5. Forsythol(isomer of forsythoside A ) 

6. rutin 

Extractum Forsythiae Siccus 

13 

7. isoforsythiaside A(isomer of forsythoside A ) 

8. forsythoside A 

99. isomer of suspensaside A 

10.isomer of suspensaside A 

11.suspensaside A 

12.Forsythin 

About 9.5% components were identified 

14 

7

Extractum Forsythiae Siccus 

still exist other components : 

Conclusion: 

� Saccharides The content of free 

monosaccharid 

and polysaccharide were low, 

most saccharides form 

glycosides 

�Flavonoids �Flavonoids Total flavonoids: 43 43.9% 9% 

�Polyphenols Total phenols: 36.7% 

Not absorbed by casein: 34.5% 

Tannin : 2.2% 

Extractum Bear Bile Powder 

ursodeoxycholic acid 

M.W.392 

UDCA and CDCA 

Chenodeoxyholic acid 

M.W.438 

Unknown compound 

80% ingredients were identified 

Solvent peak by 

gradient elution 

DAD 

15 

HR-MS 

16 

8

Substantial basis research achieve a comprehensive quality 

control of CMM 

Tanreqing Injection - standard improving 

� Flavonoids in Radix 

Scutellariae 

� Organic acids in Flos 

Lonicerae 

� Bile acids in Bear Bile 

powder 

Research Methods: 

� Fingerprint 

�Ensure uniformity and stability of the 

preparation 

� Assay: Simultaneously 

multi-components 

determination Flavonoids 

in Radix Scutellariae 

�Specific detection method adopted,70% of 

the components were confirmed (HPLC or MS) 

Fingerprint chromatograph of three extractums 

baicalin oroxylin-7-O-glucoronide 

chrysin-7-O-glucoronide 

scutellarin 

wogonoside 

cryptochlorogenic acid 

chlorogenic acid 

neochlorogenic acid 

caffeic acid 

noroxylin 

wogonin 

oroxylin A 

3,4- dicaffeoylquinic acid 


ursodeoxycholic acid chenodeoxyholic acid 

17 

� Flavonoids in Extractum 

Scutellariae Siccus 


� Organic acids in 

Extractum Flos Lonicerae 

Siccus 

most of the components have been identified. 

� Bile acids in Bear Bile 

powder 

18 

9

glc-O 

Peak identified in the fingerprint of Huangqi Injection 

O 

H O 

6 peaks can be detected both 

in the herbal medicine and its 

preparation 

Fingerprint of Huangqi Injection (including Polysorbate 80) 

OH 

OMe 

glc-O 

O 

H O 

1:calycosin-7-O-β-D-glucoside 2:Formononetin-7-O-β-D-glucoside 

glc-O 

4:Isoastragaloside 

O 

HO 

OMe 

OMe 

HO 

5:Calycosin 

O 

H O 

H 

OH 

OMe 

OMe 

glc-O 

O 

O 

OMe 

3:pterocarpan-3-O-β-D-glucoside 

HO 

6:formononetin 

We are further studied, as far as possible the full control of its quality 

O 

H O 

Chiral Mobile Phase Additives resolved the separation of the isomers 

of total glucosides in Centellae Herba 

major constituents Problems in the existing standard Solutions 

�Asiaticoside 

�Madecassoside 

�Multiple pairs of 

ursane type and 

oleanane-type 

isomers 

�As the determination method, 

UV was poor in specificity 

�The separation of 

madecassoside and its 

isomers was difficult by HPLC 

using conventional mobile 

phase 

OMe 

19 

�The multi-components 

(asiaticoside, madecassoside 

and its isomers) were 

determined simultaneously 

with the addition of βcyclodextrin 

in the mobile 

phase. 

The separation of the two isomers 

was a long-standing challenge in the 

quality control! 

chromatogram of the total glucosides in Centellae Herba by HPLC using conventional mobile 

phase 

H 

OMe 

10

The standard characteristics chromatogram of the total 

glucosides in Centellae Herba 

S1:Medecassoside C 48H 78O 20 

Madecassoside Asiaticoside 

1:Asiaticoside B C 48H 78O 20 

The two isomers have been well 

separated with our method as shown 

by peak1 and S1. 

This allowed: 

�The quantitative control of Centellae 

Herba. 

�The overall quality control including 

fingerprinting, heavy metals and 

harmful elements. 

� It may be helpful to 

consider our method 

since Centellae Herba will 

be in full control of the 

ingredients,in its to be 

issued standard by USP. 

Ⅲ Improve the quality control of toxic and effective medicine. 

Cinnabar and Realgar: Specificity examination and determination 

�Cinnabar and 

realgar belong to 

mineral medicines in 

China. 

�USP does not 

accept them as 

drugs. 

�Just talk about how 

we conduct the study 

of mineral drugs. 

Cinnabar 

Realgar 

Major components Problems in the existing standard 

�Cinnabar: mercuric 

sulfide (HgS) 

�Realgar: arsenic 

disulfide (As2S2) 

�Both of them are active 

but toxic ingredients 

�Volumetric method is used in 

the existing standard 

�Inapplicable to preparations 

analysis 

�Lack of reliable quality control 

method of Chinese patent 

medicine 

� Established the determination method for 

cinnabar and realgar 

� Solved the problem of lack of reliable 

quality control method of Chinese patent 

medicine 

� Provide a reference for more than 430 kinds 

of preparations. 

11

Source investigation 

and identification 

To define the origin 

Solutions 

Study on Standard In vivo test 

Quality control indexes 

reflecting the medical 

characteristics 

Method validation 

To explore the 

metabolism process 

References or results 

Analysis method 

of in vivo test 

Proposal of limit 

Data analysis, to explore the 

dose-effect relationship 

Established the operational 

Quality standard 

Phase analysis results of X-Ray 

Comparing with the Joint Committee on Diffraction 

Standards (JCPDS) standard cards of international 

Ch.P Standard: centre for diffraction data (MDI (MDI.Jade5) Jade5) 

Realgar: 

�As 2S 2 (main component) 

�As2O3 (limited) 

Composition of Realgar (complex): 

Cinnabar: 

Detected: 

? 

�As4S4 and AsS (main component) 

�HgS (main component) �As2S3 and SiO2 (trace component) 

Not detected: 

�As �As2O 2O3(toxic 3(toxic component) 

�As2S2(pharmacopoeia component) 

23 

Composition of Cinnabar 

(simple): 

HgS(main component) 

The results indicate: The main component of Realgar is different from the one in 

state standard. The result is to be confirmed and amendments is to be proposed. 

24 

12

Frame of the quality standards study 

Main components p 

Determination of 

principal components 

�Volumetric 

�Volumetric 

method 

�F-AAS 

�ICP-OES 

Main components related to Toxic associated 

effectiveness and toxicity elements 


soluble As and Hg 

�ICP-MS 

�HPLC-ICP-MS 

Determination of As 

and Hg species 

Analysis of the main components 

Methods 

�Volumetric method 

�F-AAS 

�ICP-OES 

�Why ICP-MS is not 

chosen 

� The measuring solution need 

multiple dilutions for the high 

sensitivity of the instrument 

�The instrument is in the risk of 

being contaminated for the high 

content of mercury/arsenic in 

samples 

Results 


Fe and Pb 

�Fe: F-AAS 

�Pb:GF-AAS 

�Crude drugs 

Results of volumetric method, F- 

AAS and ICP-OES are consistent. 

Volumetric method can be used 

for crude drugs(Pharmacopoeia 

method) 

�preparations 

Volumetric method can not 

eliminate interference. 

Instrumental method have to be 

used. 

25 

26 

13

The method development of soluble arsenic 

/mercury analysis and their speciation 

Problems: 

�How to simulate the body environment using 

appropriate in vitro method? 

�How to make the method more reproducible? 

�How to explain Cinnabar and Realgar g conforming g to 

regulations? 

More difficult ,exceeding the expectation 

The focal points influencing the reproducibility of 

the results? 

Focal points 

� Granularity 

Samples come from 

�Crude drugs: 

Realgar, 12 batches 

Cinnabar,14 batches 

� Solvent and ways of 

�Processed samples: 

extraction 

Processed by factory of 

professional TCM and herbal 

� WWays of f purification ifi ti 

First 

slices company using the crude 

drugs 

�Preparations : 

Produced by TCM company using 

the crude drug and processed drugs. 

study on the same batch of mineral medicine, 

processed, and its preparations 

28 

27 

14

�Extracting way 

The factors influencing the analysis 

Factors Degree of influence 

�Extracting solvent 

�Sample amount 

�Extracting temperature 

�Obvious 

�Significant 

�Not influencing 

�Not distinctive 

�Extracting time 

�Not distinctive 

�Purification �Purification way �Veryy significant g 

Purification way is the priority among these 

factors 

Factors affecting the analysis, 

and method optimizing 

Difficulties Solutions 

Many factors affecting the reproducibility 

�Granularity:affecting surface area, the extent of 

dissolution, thereby influencing the concentration. 

�Extraction:The process of dissolution of arsenic and 

mercury is constant. Purpose of the experiment is 

to simulate over the maximum amount in vivo. 

�Purification:There are three existence forms of 

soluble arsenic and mercury: small suspended particles, 

complexes, and ionic forms. Any small changes from the 

way of purification will directly affect the determination 

results. 

�To homogenize the particles 

�To choose a appropriate 

extraction method to simulate over 

the maximum amount in vivo. 

29 

�To �To choose an appropriate purification 

Method, making the results of soluble 

arsenic and mercury or their species more 

reproducible 

30 

15

Factors affecting the analysis,and method 

optimizing——Conclusions 

Realgar and processed Realgar: Cinnabar and processed Cinnabar: 

�The amount of the soluble 

species of arsenic are equivalent. 

�The soluble arsenic, or the species 

of arsenic could be taken as the 

standard index. 

�Soluble mercury are represented with 

species of mercury for the moment 

�Further study is needed on analyzing of 

soluble mercury 

Preparations with Realgar and Cinnabar as components: 

�The total amount of soluble arsenic and the amount of species 

of arsenic are equivalent in the preparations. 

�The influence on the soluble arsenic/mercury and the species 

varies in different preparations, so the standards of each 

preparations need to be studied respectively. 

HPLC-ICP-MS Chromatograms of arsenic and mercury mixed species standards 

Six species of arsenic: AsC—Arsenocholine、AsB—Arsenobetaine、As(Ⅲ)—Arsenious 

acid、 DMA—Dimethylarsinate、MMA—Monomethylarsonate、As(Ⅴ)—Arsenic acid 

Three species of mercury: Hg(Ⅱ)、 MeHg—Methylmercury、 EtHg— 

Ethylmercury 

31 

32 

16

RRealgar l 

Cinnabar 

HPLC-ICP-MS Chromatograms of species in Reagar and Cinnabar 

Single oral dose 

administration 

Multiple oral dose 

administration 

Urine 

Feces 

Only As(Ⅲ) andAs(Ⅴ) were found 

Only Hg(Ⅱ) was found 

Excretion 

In vivo test 

Absorption 

Realgar 

Cinnabar 

Distribution Accumulation 

The research process and preliminary results in vivo : 

�To study the relation of in vitro test and in vivo test 

�To study the distribution and accumulation of arsenic/mercury in 

organs 

�To study the relation between accumulation and the toxicity 

34 

33 

17

content(%) 

8 

6 

4 

2 

0 

Study on correlation between in vitro test and the acute 

toxicity test in mice 

Investigate the correlation between index components of realgar and LD50 of mice 

because of the acute toxicity of realgar. 

y = -2.7203x + 7.1463 

R 2 = 0.9718 

y = -3.0448x + 9.7306 

R 2 = 0.9398 

1 1.2 1.4 1.6 1.8 2 2.2 

LD50(g/kg) 

可溶性砷 

价态砷 

线性 (可溶性砷) 

线性 (价态砷) 

�There is negative correlation 

between LD50 and soluble 

arsenic, species of arsenic 

�Correlation coefficient are – 

0.9694 and -0.9858 

Study on correlation between in vitro test and the 

acute toxicity test in mice 

The results indicate: 

�There is good correlation between LD50 and the amount of soluble 

arsenic and species of arsenic, and light correlation between LD50 

and the total arsenic, it indicate that the acute toxicity cause mainly by 

soluble arsenic and species of arsenic. 

�The contents of soluble arsenic and species of arsenic of realgar in 

same batch increase after processing. It improve the efficacy while 

increase the risk of poisoning. 

�The dates of in vitro test can effectively reflect the toxicity of realgar. 

18

Correlation between in vitro and in vivo test 

Content of arsenic and mercury at 1h in tissues, organs and urine after 

single g oral dose administration 

�Cinnabar:The amount of species of mercury in vivo test is 19% of that in vitro test. 

�Realgar: The amount of species of arsenic in vivo test is 74% of that in vitro test. 

The results indicate that: 

�I �In vitro i test could ld simulate i l the h maximum i amount obtained b i d in i vivo i test to a 

certain extent. 

�The amount of species of mercury is less than the species of arsenic, so acute 

toxicity of Cinnabar occurs less likely than Realgar, which is in accordance with 

the reported conclusion. 

The future of quality control of CMM 

FULL 

SAFE 

EFFECTIVE 

CONTROLLABLE 

37 

19

Thank you for your attention! 

Your suggestion is welcome! 

39 

20

Combating Counterfeiting and Substandard 

Medicines in Developing 

Countries 

PPatrick t i k LLukulay, k l Ph.D., Ph D Di Director t 

Promoting the Quality of Medicines Program 

United States Pharmacopeia, USA 

Bad Medicines 

� When hygiene and nutrition fail to prevent disease 

Pharmaceuticals 

► Delayed treatment 

► Mortality 

► Poverty 

► Resistance 

11/29/2010 

1

Origins and Spread of Drug Chloroquine Resistance 

Defining the Problem 

�Unregulated market 

�Un-informed public 

Plowe CV. The evolution of drug-resistant 

malaria. Trans R Soc Trop Med Hyg. 11 

Dec 2008. 

Wellems lab/NIH NIAID 

�Undue focus on Price than quality 

11/29/2010 

2

Good Legislation and Regulation 

Unregulated— 

Business owner 

diagnoses, prescribes, 

and dispenses 

The Patient’s Protective Shield 

Regulated— 

Doctor or health care worker 

diagnoses g and p prescribes, , 

pharmacist dispenses 

Manufacturer Wholesaler/Pharmacy 

Regulator 

Patient 

m2 

11/29/2010 

3

Slide 6 

m2 This slide is set to transition in pieces: photos, thenclip art. Is that how you intended? 

mlf, 8/31/2010

The Regulatory Challenge 

Poor Registration Practices 

• Backlog of dossiers 

• Porous borders, high tariffs 

• High staff turn-over- political or economic 

Poor Post-market surveillance 

• Un-controlled market and poor pharmacovigilance 

Poor enforcement action 

• Weak legislation- no regulation without legislation 

• Weak penalties 

• Political will to enforce the law 

QAMSA Study 

The Quality of Antimalarials in Sub-Saharan Africa 

(QAMSA) Study 

► Cameroon 

► Ghana 

► Ethiopia 

► Kenya 

► Madagascar 

► Malawi 

► Nigeria 

► Senegal 

► Tanzania 

► Uganda 

Artemisinin-based Combination Therapy (ACT) 

Medicines and Sulfadoxine-Pyrimethamine (SP) 

11/29/2010 

4

Artemesinin Compound 

QAMSA Study 

Artemesinin 

Figure 1. Quality of Antimalarial Medicines Sampled from 

Per rcentage of Total Samples Samples Samples Samples Tested 

100% 

90% 

80% 

70% 

60% 

50% 

40% 

30% 

20% 

10% 

70 

30 

74 

26 

56 

44 

0% 

Madagascar Uganda Senegal 

Pass, % 

Fail, % 

11/29/2010 

5

Key Findings 

Nummber 

of Samples 

16 

14 

12 

10 

8 

6 

4 

2 

0 

Figure 8. Quality of Antimalarial Medicines Sampled from Different 

Distribution Sectors in Senegal 

2 

8 

11 

4 

Pass 

Fail 

1 

1 

Informal Private Public Informal Private Public 

SPs ACTs 

Failed Samples in Ghana 

Sentinel Site 

Origin of Drug Sample 

Name of Facility 

Medicine 

Name 

Active Pharmaceutical 

Ingredient(s) 

Basic Overall Test 

Conclusion 

Tarkwa Chemical Store 

Tarkwa 

Bolga 

Bolga 

Kumasi 

Private Pharmacyy 

Private pharmacy 

Health Drug Stores 

Chem. Store 

Trafan 

Sulfadoxine & 

Pyrimethamine 

Failed ldDissolution l 

(5% Release after 

1 hr) 

Ho Health Services 

Tarkwa Jemoz Pharmacy 

Tarkwa 

Tarkwa 

Kumasi 

Millenium Pharmacy 

Nat & Sons Pharmacy 

C. Crentsil Pharmacy 

Tab Metakelfin 

Sulfamethopyrazine & 

Pyrimethamine 

Counterfeit 

Bolga Superlite Pharmacy 

Tarkwa Kekule Pharmacy Artesunate Artesunate Counterfeit 

Ho 

Sape Agbo Memorial 

Hospital 

Quinine 

Sulfate 

Quinine Sulfate Counterfeit 

Kumasi Private Pharmacy 

Kumasi Health Centre 

Malmed Artesunate & Amodiaquine Failed Dissolution 

Kumasi Private Pharmacy 

Accra Private Pharmacy 

Phamonate‐ 

400 

Artesunate & Amodiaquine Failed Dissolution 

4 

5 

9 

7 

8 

2 

11/29/2010 

6

Medicines are not ordinary commodities 

Stability of ACTs 

Metabolize to 

Artemesinin Dihydroartemesinin 

Hydrolysis Product 

11/29/2010 

7

Addressing the Problem 

�Regulated Market 

�A �An IInformed f d public bli 

�Focus on Quality 

Post market Surveillance 

Verify what is out there 

� Quality status 

� Registration status of medicines 

� Adhere to treatment guidelinese.g. 

monotherapy versus FDC for malaria 

� Leakages from the public sector 

� Expired medicines 

�� Medicines availability data 

11/29/2010 

8

PMS–Testing Methods Used in Field 

GPHF Minilab ® 

Everything needed for drug testing fits into two 

transportable units units, each about the size of a 

suitcase and weighing about 40 kg 

Basic Tests for the GPHF Minilab® 

Visual Inspection Disintegration Test Colour Reactions Thin Layer 

Chromatography 

Physical Inspection Release Rate 

Identification ID, Assay & Imp 

11/29/2010 

9

Advantages of the Minilab 

� Requires less training- addresses human resource 

issues and rapid staff turn over 

�� Task shifting 

� Low maintenance cost- address inadequate resources 

� Rapid test results—allows quick regulatory action 

� Portable- allows the decentralization and ownership of quality 

control. Deployed in the provinces and remote places 

� Need little infrastructure—battery powered devices 

PQM–Two Step Approach 

Screening 

Confirmatory Testing 

11/29/2010 

10

Case Study #2: Ghana 

� Sites selected based on the following criteria: 

� Epidemiological 

� GGeographical; hi l 

Administrative 

� Areas known for 

traffic in fake drugs 

� Border provinces 

Source: USP, 2009 

Case Study #3: Cambodia 

� MQA Improvement since 2003 

I. Lab strengthened 

II II. PPostmarketing t k ti surveillance ill (PMS) (PMS), 

with enforcement 

III. Active public awareness campaign, 

e.g., PSA & videos 

� PMS Results 

�� 932 AML samples collected 2003 2003-08 08 

� 2003-2004: 23% & 27% failure rates 

� 2006-2007: 11% & 8% failure rates 

Source: USP, 2009 

Fake 

Genuine 

11/29/2010 

11

Conclusion 

�Quality is not a luxury but a necessity 

�Access to good quality medicines is 

a right not a privilege 

�Provision of commodities must be 

matched with services and measures 

to ensure quality 

�Conditio sine qua non: Political Commitment 

Substandard Meds: A Health Threat First and Foremost 

“ 

Dying from a disease 

is sometimes 

unavoidable, dying 

from a medicine is 

unacceptable. 

” 

-- V. Lepakhin, Geneva 2005 

From “The need for pharmacovigilance,” presented by Mary R. Couper and 

Shanthi Pal at the WHO Technical Briefing Seminar, September 24, 2009 

11/29/2010 

12

PQM Staff 

Where we’re from: Argentina, Cambodia, 

Democratic Republic of the Congo, 

Ethiopia, Ghana, Jamaica, Laos, 

Mexico, Morocco, Philippines, Russia, 

Sierra Leone, Switzerland, United 

States, Zambia 

Languages we speak: Akan, 

Arabic, Cambodian (Khmer), 

Chinese, English, French, 

German, Hebrew, Krio, Lao, 

Mende, Portuguese, 

Spanish & Russian 

11/29/2010 

13

11/29/2010 

14

Day 1 - US Pharmacopeial Convention

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