Impurity Profiles and Degradation Products in Raw …...Some of the biggest culprits: the quality of...

©2013 Waters Corporation 1

Impurity Profiles and Degradation Products in Raw Materials and Pharmaceutical Products

Michael D. Jones

Ignatius (Iggy) Kass, Ph.D.

Sr. Field Marketing Manager

www.waters.com

Confidential


Content

Understanding your Challenges:

– Risks of impurities in pharmaceuticals

Strategies for Impurity Profiling

– Quality Control, batch acceptance

– “Impurity Profiling”

Application examples

Summary

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The risk of impurities

Unwanted chemicals influence the efficacy and safety of the pharmaceutical products.1

– “Any component of the drug product that is not the chemical entity defined as the drug substance or an excipient in the drug product.”2 (ICH Q6A: Specifications).

– An excipient is a pharmacologically inactive substance formulated with the active ingredient ("API") of a medication

Impurity profiling (i.e., the identity as well as the quantity of impurity in the pharmaceuticals) is receiving important critical attention from regulatory authorities.1

Various regulatory authorities like ICH (International Conference on Harmonization), USFDA, Canadian Drug and Health Agency are emphasizing on the purity requirements and the identification of impurities in Active Pharmaceutical Ingredient’s (API’s).1

1. Tegeli et.al, IJDFR volume 2 Issue 4, Jul-Aug.2011

2. ICH Q6A: Specifications

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Regulations to promote good science Impurity Thresholds for Drug Substances

Detect

– Know that it is there

Identify

– Know what it is

Qualify

– Know that it is safe

Maximum Daily Dose

Threshold

Reporting Identification Qualification

≤ 2 g/day 0.05%

0.10% or

1.0 mg/day intake (lower)

0.15% or

1.0 mg/day intake (lower)

> 2 g/day 0.03% 0.05% 0.05%

Guidance for Industry, Q3A Impurities in New Drug Substances, February 2003; \\CDS029\CDERGUID\4164fnl.doc Draft Guidance for Industry, ANDAs: Impurities in Drug Substances, January 2005; J:!GUIDANC\6422dft.doc

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Impurity Profiling Business Impact on Operations

Acceptance or rejection of raw material for production

– Impurity profiles define material acceptance or rejection

– Out of specification (OOS) : Reject raw materials

Sign-off of finished API or drug product for release

– Ensures quality of the product

– Impacts time to market and customer satisfaction

Adhere to cGMPs and CMC regulatory requirements

– Regulatory filings dependent on time to complete studies

– Results must be accessible and confidently defendable to regulatory

authorities

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Analytical Challenges

Throughput – Multiple batches are required to be analyzed

– Shorter turn round times to increase profitability

Sensitivity – Low level impurities can pose a serious issues with genotoxicity

– Sample impurity ”finger print’ used to support counterfeit activities

Specificity – Ability to quickly detect drug or excipient related material simplifies

impurity detection and identification

Ease of collecting required data – Transfer procedures within and between labs

– Employee turnover

Informatics – Getting results that drive decisions

– in silico tools (modeling, prediction) utilize prior knowledge to allow analytst to focus on the more challenging tasks

– Merging data from different sources (experiments, labs, geographies, etc)

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©2013 Waters Corporation 7 Michael D. Jones, PBO

Potential Impurities

Degradants (Forced degradation studies)

– Acid, base, heat, light, etc.

Synthetic intermediates

In process 2007 Viracept recall, Roche Pharmaceuticals, ethyl mesylate, genotoxic. - Tracked

down to a reaction between the active ingredient and chemicals used in Cleaning in

Manufacturing.

Genotoxins

And anything else……

“Alerting functional moiety” that is shared with the impurity or API structure

- At any level

Muller, L., Mauthe, R.J., 2006. A rationale for determining, testing and controlling specific impurities in pharmaceuticals that possess

potential for genotoxicity. Regulatory Toxicology and Pharmacology 44, 198-211

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Sensitivity Required for Genotoxins

Impurities should be reported at levels in excess of the 0.03 – 0.05% (300 – 500 ppm, by weight)

range according to ICH_Q3A_R2 guidelines. Genotoxic impurities, or potentially genotoxic impurities,

must be controlled at levels significantly lower than this. Typically, developing limit tests of less than

50 ppm for highly toxic impurities is readily achievable. However it can be difficult to develop a test

to control a particular genotoxic impurity at the current EMEA guideline limit of 1.5 ppm (0.00015%

w/w) or less.

– AAL Website

"For genotoxic impurities we need very sensitive and selective methods. One needs higher

sensitivity to determine ppm-level impurities and selective methods to separate low levels of

genotoxic impurities from base line noise and other organic impurities. The typical HPLC methods with

a nonspecific detector (e.g., UV) that are used to measure organic impurities may not be appropriate

to quantitate low ppm levels of genotoxic impurities.“

– Maribel Rios, Pharmaceutical Technology

– 10 – 100 times more sensitivity needed

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The safety and business risk even if you meet regulations

Some of the biggest culprits: the quality of raw materials, faulty labeling

and packaging and contamination.1

A number of factors are fueling the recall surge. The stampede by

drugmakers to be first to bring generic versions to market, after drugs lose

patent protection, is one of them.1

– "The first applicant typically gets the lion's share of the business for the new drug,"

said Cox.

– In their hurry, drugmakers sometimes fail to spend enough time learning how best

to make the drug.1

2011 J&J Recalls helped push net income down 12 percent ($900 million) 2

1 http://money.cnn.com/2010/08/16/news/companies/drug_recall_surge/index.htm 2 http://www.bloomberg.com/news/2011-01-25/j-j-profit-declines-12-on-product-recalls-2011-outlook-trails-forecast.html 3Tablets & Capsules 2012

1 3

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Strategies

I. Quality Control

– Impurities are well defined: USP methods and standards

II. Impurity Profiling

– Full characterization: Define what may be present

– Where to begin?

• Synthetic routes – In process impurities

• Degradation – Heat, Light, Acid/base...

• Previous production on manufacturing line

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I. QC testing WATERS UPLC SOLUTION

Quick, easy and cost effective

– Typically LC/UV

– Impurity profile is already known at this point

o Information you have is the compounds retention time..

HPLC

HPLC

UPLC

UPLC

Speed

Resolution

Sensitivity

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QC testing

Fast, easy and cost effective

– Typically LC/UV

– Impurity profile is already know at this point


Example

USP method and known impurities

API Related

Known Impurities

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QC testing


– Typically LC/UV



Example


API

Known Impurities

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QC testing


– Typically LC/UV



Example


API

Known Impurities

What is it?

?

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Time

%

Potential Risks

API

Known Impurities

?

What if we look at these peaks by Mass Spectrometry?

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1: MS gives an extra dimension of separation:

210 350nm

100

%

210 350nm

100

%

210 350nm

100

%

200 300 400m/z

100

%309.1

311.1

200 300 400m/z

100

%

309.1

311.1287.1

200 300 400m/z

100

% 309.1

287.1

Mass Spectrometry (Single Quadrupole)

Time

%

API

Known Impurities

?

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What does Contamination look like? Supplier Comparisons: Budesonide

Supplier A

Supplier B

Supplier C-Lot1

Supplier C-Lot2

Representative MS TIC Chromatograms

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II. Waters Solution for Complete Impurity Profiling

UPLC – Speed of analysis

– Challenging separations of related compounds

o Peak Capacity

Dynamic Range – Sensitivity for low level peaks

– Mass Measurement Accuracy for High Intensity peaks

Informatics – Simplicity of processing / Setup

– Easy operation for known analytes

– Detection of unknowns o Automatic calculation of elemental composition

o Confirmation / Elucidation with MSE data sets

– Correlation of PDA Data in reporting

Detect

– know that it is there

Identify

– Know what it is

Qualify

– Know that it is safe

– (Biology and safety

studies)

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Summary of Major Simvastatin Degradation Products*

MSUVMSUVMSUVMSUVMSUV

3.970

4.423

5.209

5.254

5.699

6.222

6.322

6.881

7.257

3.862

3.841

3.594

3.449

3.137

3.107

2.986

2.938

2.397

2.308

2.191

2.161

2.116

2.062

1.926

1.886

1.760

1.699

Photo DegradationPeroxide

Oxidation

Temperature

Degradation

Base HydrolysisAcid Hydrolysis

Peak

Rt (min)MSUVMSUVMSUVMSUVMSUV

3.970

4.423

5.209

5.254

5.699

6.222

6.322

6.881

7.257

3.862

3.841

3.594

3.449

3.137

3.107

2.986

2.938

2.397

2.308

2.191

2.161

2.116

2.062

1.926

1.886

1.760

1.699

Photo DegradationPeroxide

Oxidation

Temperature

Degradation

Base HydrolysisAcid Hydrolysis

Peak

Rt (min)

*Does not include impurities detected in Simvastatin standard.

MS Detection is VERY Sensitive, But still a Complementary Technique

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Typical Workflow

Flag questionable impurities

Isolate impurities by API removal or impurity enrichment

Perform MS/MS or MSn structural studies

Confirm by NMR

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Design a Waters Strategy

Separate A well developed impurity

profile method is KEY

Identify MSE

Metabolynx XS

Elucidate MassFragment

Isolate Mass Directed Autopurification

Scalable Chemistries

?

C4H7N2O3

NN

S

N

O

OH

Man

ag

e

Compile

Track

File

NuGenesis SDMS

NuGenesis SDMS

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Method Development

Selectivity [α]

Stationary Phase

Organic Modifier

Mobile Phase pH

Column Manager

•Stack to support up to 6 columns

•Independent heating/cooling zones

Sample Manager—FTN

•Direct injection simplifies mode of injection selection

•Injection volume flexibility without reconfiguring injector

Quaternary Solvent Manager with Optional Solvent Select Valve

•Up to 9 solvents with integrated solvent selection valve on the D line (1-6 solvents)

•Select study variables

•Define study ranges Experiment

Design

•Conduct formal experimental design

•Analyze results, build equations of variable effects

Knowledge Space

•Define best conditions

•Define robustness

Design Space

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Impurity ID/Elucidation

Generic methodologies still provide maximum coverage Automated data interpretation Identify drug related compounds Elemental Composition /Structural elucidation

The most sensitive, exact-mass,

quantitative and qualitative benchtop

MS system

Uses UPLC/MSE, a simple, patented

method of data acquisition to

comprehensively catalog complex

samples in a single analysis

Provides the most complete system

solutions backed by superior support to

ensure customer success

Guarantees maximum system

performance and usability through our

implementation of Engineered

SimplicityTM

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Identification …detection in complex mixtures

Intensity 6.04 x 106

Intensity 535

Mass Measurement Accuracy and Quantitative Dynamic Range Concentration of drug very high Excipients add to complexity (PEG, etc.)

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Alternate Scanning LC-MS (LC-MSE) …time resolved mass measurements Parallel LCMS (LCMSE)

…a dual function LC-MS protocol

MSE

is a UNBIASED process

MSE

is a CONTINUOUS process

1 sec

LE

EE

LE

EE

EE EE

D.

m

All ions all the time

Very Simple Setup

Bateman et al, Anal Chem (2002) Silva et al, MCP (2005) *Patented technology

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Identification with MSMS Example: Nefafzodone

N

N

N

O

CH3

ON

N

Cl

N

N

N

O

CH3

ON

N

Cl

NCH3

N

N

O

CH3

N

N

Cl

N

N

N

O

CH3

OCH3

CH3N

N

Cl

NH

N

N

O

CH3

CH3

MS (Detect)

MSMS (Know what it is) Confidential


Systematic Bond Disconnection and Exact Mass

MassFragment Software does this automatically

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Parent drug fragment ion characterization with MassFragment

Nefazodone

Nefazodone fragments

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Impurity Isolation for alternate techniques (NMR, Optical, etc..)

Mass directed purification offers

far greater specificity and

efficiency than traditional UV

directed systems

With both analytical and

preparative performance available

as standard, method

development, purification and

fraction analysis are performed

automatically, and without

compromise

OpenAccess software provides

access to the advanced fraction

collection functionality by even

the most novice users

Crude mixture

? LC/MS

purification

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Key Contributors

Michael D Jones

Warren Potts

Marian Twohig

Confidential


Test Case Compound

Quetiapine fumarate (Anti-Depressant)

Chemical Name:- Bis[2-(2-[4-(dibenzo[b,f][1,4]-thiazepin-11-

yl)piperazin-1-yl] ethoxy) ethanol] fumarate

Quetiapine fumarate has no chiral centers and only one morphological

entity has been detected throughout development

N

N

S

N O

OH

Quetiapine

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Test Case 1: Qualitative Profiling Management Confirm Known Impurities

A Workflow Approach for the Identification and

Structural Elucidation of Impurities of Quetiapine

Hemifumarate Drug Substance

Increase Visualization using Metabolynx w/ MassFragment

Search for API Relationships

Analyze using MSE

Fragment Ion Analysis

*Optional

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Quetiapine Impurity Profile

Michael D. Jones

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MetaboLynx XS Browser

Impurity Results Table

Corresponding impurity spectra

Combined impurity trace

Extracted impurity ion trace

Control v. Analyte Known v. Unknowns

Chromatography data

Exact Mass data Apply Mass

Defect Filters Central

Repository Confidential


Rapidly Lists Detected Peaks:

Automatically Generates a List of Impurities within Minutes Confidential


Quetiapine Impurity Profile has many UNKNOWN Peaks

Michael D. Jones

Quetiapine Confidential


One Quetiapine Impurity MSE Result

Michael D. Jones

Com

mon F

ragm

ent

Ions t

o A

PI

• 279.1030

• 253.1522

• 221.1132

• 210.0520

• 183.0358

• 139.0473 MSE spectra of Quetiapine Impurity [M+H] = 340.1536

Use MSE data to identify structural components you may already know!

N

N

S

N O

OH

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MassFragment Impurity Investigation of m/z 456.2305

OH

CH3

CH3

N

N

S

N

O

O

MassFragment summary report

Michael D. Jones

MSMS spectrum

MOL file used for [M+H] = 456.2305

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FRAGMENT ANALYSIS

Michael D. Jones

Data Mines Known v. Unknowns

Precursor & Product Ions

Review XIC in Browser

Links to MassFragment

High - CE scan window

Expected/unexpected impurities

Chromatographic common precursors of selected ion

Rapidly Identify Related Impurities

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Test Case2 : Targeted Identification and Isolation of a Previously Unidentified Impurity

Identification and Characterization of an Isolated

Impurity Fraction: Analysis of Impurity 402 found in

Quetiapine Fumarate

Elucidate with MassFragment

Generate Elecomp and proposed structure

Analyze Fraction by MSE

Scale Methodology for Preparative Isolation

Identify Unknown

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UPLC Impurity Profile Method: Acid Degradation of Quetiapine

Quetiapine

Major Unknown

impurity LC System: Waters® ACQUITY UPLC® System

Column: ACQUITY UPLC BEH C18 Column

2.1 x 100 mm, 1.7 µm

Column Temp: 65 ˚C

Flow Rate: 800 µL/min.

Mobile Phase A: 10mM Ammonium Bicarbonate,

pH 9.0

Mobile Phase B: Acetonitrile

Gradient: 15-39%B for 10.5min,

39-43%B to 14.4minutes,

43-95%B to 18min,

hold at 95% for a total run time of 20min

1.326 Peak 1 - SQ 1: MS Scan 1: 50.00-600.00 ES+, Centroid, CV=Tune

402.1

Inte

nsit

y

0.0

4.0x106

8.0x106

1.2x107

1.6x107

m/z110.00 220.00 330.00 440.00 550.00

m/z=402

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Transfer from UPLC to Preparatory

UPLC Column 2.1 x 50 mm,

1.7µm

Time (min)

Flow Rate (mL/min)

%A %B Segment Duration

Time (min)

Segment Duration

(c.v)

0.0 0.8 95 5 0 0

2.0 0.8 40 60 2 9.25

3.0 0.8 5 95 1 4.62

Prep Column 19 x 100 mm,

5 µm

Time (min)

Flow Rate (mL/min)

%A %B Segment Duration

Time (min)

Segment Duration

(c.v)

0.0 25 95 5 0 0

10.5 25 40 60 10.5 9.25

15.7 25 5 95 5.2 4.62

Calculated Migration to Prep from UPLC

Diehl, Diane M. et al. Method Migration From UPLC Technology to Preparative HPLC, Waters App Note 720002375EN

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Mass Directed Auto-Purification

basic conditions

Time6.00 7.00 8.00 9.00 10.00 11.00

%

0

100

Que_Aq_Only_Acid_Deg_22 1: Scan ES+ TIC

2.40e8

Pump 2545 Binary Gradient Module

Inject/Collect 2767 Sample Manager

Detector 2998 Photodiode Array

Mass Spec 3100 Mass Detector

Column 19 X 100mm XBridge, 5 µm

Solvent A 10 mm Ammonium

Bicarbonate, pH 9

Solvent B Acetonitrile

Flow Rate 25 mL/min

Gradient 5% - 60%B over 10.5 min.

flushed for approx 5 minutes

95% organic

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Verification of Pooled m/z 402 Fractions

1.3

25

Inten

sity

0.0

6.0x106

1.2x107

1.8x107

2.4x107

Minutes0.60 1.20 1.80 2.40 3.00

AU

0.000

0.003

0.006

0.009

0.012

Minutes0.00 0.60 1.20 1.80 2.40 3.00

1.309 Peak 1

302.3

379.2 396.0

AU

0.000

0.010

0.020

0.030

0.040

nm240.00 280.00 320.00 360.00

ES+ TIC UV 250nm

1.326 Peak 1 - SQ 1: MS Scan 1: 50.00-600.00 ES+, Centroid, CV=Tune

402.1

Inte

nsit

y

0.0

4.0x106

8.0x106

1.2x107

1.6x107

m/z110.00 220.00 330.00 440.00 550.00

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Elemental Composition

Molecular

Formulas

Mass Error

Values

i-Fit

Settings

Flexibility

Elecomp

Provides

Elemental Composition report for quetiapine impurity with an observed accurate mass of m/z 402.1838.

= Quetiapine + H2O

Confidential


Possible Structures

O

NNH

S

N

O

OH

O

N

NO

OH

N

S

O

N

NH2

S N

O

OH

S-oxide and C=N reduction based structure?

C21 H28 N3 O3 S

C=N cleavage creating a Nitroso based structure?

C21 H28 N3 O3 S

C=N cleavage creating an Amino/ketone based

structure?

C21 H28 N3 O3 S

S

N

N

N

O

OH

?

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Ruling out Proposed Structure “B” Nitroso- based compound

O

N

NO

OH

N

S

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Ruling Out Proposed Structure “A” S-oxide- based compound

O

NNH

S

N

O

OHConfidential


MassFragment

O NNH2

S

N

O

OH

[M+H] = 402

Report based on:

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Accurate Mass MS/MS

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13 C NMR (From Collected Fractions)

O NNH2

S

N

O

OHConfidential


H-NMR (From Collected Fractions)

O NNH2

S

N

O

OH

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Conclusions

Chromatographic separation and retention time reproducibility is critical to imparting confidence in the quality of your products. Some laboratories are implementing simple ms detection to minimize risk

Accurate Mass - Mass Spectrometry adds another dimension of identification for routine screening and plays a critical role in defining impurity profiles

Often scale-up is required to utilize less sensitive confirmatory analysis

Informatics that incorporate chemical know-how increase productivity by providing a route to fast and accurate reporting

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Key advantages of Waters Systems

Comprehensively catalogue complex samples in a single

analysis with consistency, specificity and wide dynamic range

with innovative UPLC and MSE technology

Engineered simplicity and IntelliStart makes exact mass

routine.

Informatics offering an automated, structure driven, chemically

intelligent workflow for complete data set reporting

Providing services bridging workflows across organizations

throughout from preclinical to final marketing

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?

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References

SIGNIFICANCE OF IMPURITY PROFILING: A REVIEW

V.S. Tegeli*, G.B. Gajeli, G.K. Chougule, Y.S. Thorat, U.S. Shivsharan, , S.T. Kumbhar

, DSTSM’s College of Pharmacy, Solapur., Maharashtra, India. 413003

– Tegeli et.al, IJDFR volume 2 Issue 4, Jul-Aug.2011

Recent trends in the impurity profile of pharmaceuticals

– Kavita Pilaniya, Harish K. Chandrawanshi,1 Urmila Pilaniya,1 Pooja Manchandani,2 Pratishtha Jain,3 and

Nitin Singh3

– J Adv Pharm Technol Res. 2010 Jul-Sep; 1(3): 302–310.

Confidential

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Impurity Profiles and Degradation Products in Raw …...Some of the biggest culprits: the quality of...

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