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Dionex products are designed, developed, and manufactured under an ISO 9001 Quality System.

Passion. Power. Productivity.

Cobra, DCMSLink, Integral, Reagent-Free, RFIC, SmartPeaks, Trinity, and ultra

are trademarks and Acclaim, CarboPac, CAD, Chromeleon, Corona, IonPac,

OmniPac, and UltiMate are registered trademarks of Dionex Corporation.

MICRO is a registered trademark of International Products Corp.

Xcalibur is a registered trademark of Thermo Fisher Scientific, Inc.

Analyst is registered tradmark of Applied Biosystems.

HyStar is a trademark of Bruker Daltonics.

© 2010 Dionex Corporation

LPN 2499 PDF 05/10 Printed in U.S.A.

Related Dionex Applications LiteratureDionex has an extensive library of methods and techniques for determining a wide variety of analytes important to the pharmaceutical industry. For more information, visit www.dionex.com and click on Life Sciences–Pharmaceuticals on the sidebar under Applications. Below is a selected list of pharmaceutical applications also available for download by clicking on Literature and Manuals under Training and Support on the sidebar, or by contacting your local Dionex representative.

PhARMACeuTICAL LITeRATuRe

Class Application AN

AntibioticsDetermination of Tobramycin and Impurities Using HPAE-PAD(see also AU 167: Determination of Tobramycin in Crude and In-Process Production Samples During Manufacturing Using HPAE-IPAD)

61

Antibiotics Determination of Neomycin B and Impurities Using HPAE-IPAD 66

Antibiotics Determination of Sulfur-Containing Antibiotics Using Integrated Pulsed Amperometric Detection 132

Antibiotics Determination of Streptomycin and Impurities Using HPAE-PAD 181

Antibiotics Analysis of Paromomycin by HPAE-IPAD 186

AntibioticsDetermination of Sulfate Counterion and Anionic Impurities in Aminoglycoside Drug Substances by Ion Chromatography with Suppressed Conductivity

190

Antibiotics Determination of N-Methylpyrrolidine in Cefepime Using a Reagent-Free Ion Chromatography System 199

Antibiotics Determination of Cefepime and Cefepime-Related Substances Using HPLC with UV Detection 205

Drugs & Metabolites Determination of Nevirapine Using HPLC with UV Detection 180

Drugs & Metabolites Determination of Carbachol In Ophthalmic Solutions Using a Reagent-Free Ion Chromatography System 194

Drugs & Metabolites Determination of Verapamil Hydrochloride Purity Using the Acclaim PA Column 195

Drugs & Metabolites Determination of Glucosamine in Dietary Supplements Using HPAE-PAD 197

Drugs & MetabolitesDetermination of Urea and Allantoin in Cosmetics Using the Acclaim Mixed-Mode HILIC Column

198

Drugs & MetabolitesDetermination of Galactosamine Containing Organic Impurities in Heparin by HPAE-PAD Using the CarboPac PA20 Column

233

Drugs & MetabolitesDetermination of Oversulfated Chondroitin Sulfate and Dermatan Sulfate in Heparin Sodium Using Anion-Exchange Chromatography with UV Detection

235

Drugs & Metabolites Extraction of Nitroglycerin from Transdermal Patches by Accelerated Solvent Extraction 327

Drugs & Metabolites Analysis of Sumatriptan in Cerebro-spinal Fluid Using Capillary LC/MS/MS 504

Traditional Chinese Medicines

Determination of Anthraquinones and Stilbenes in Giant Knotweed Rhizome by HPLC with UV Detection

232

Anions & Cations Determination of Trifluoroacetic Acid (TFA) in Peptides 115

Anions & Cations Quantification of Anions in Pharmaceuticals 116

Anions & Cations Determination of Inorganic Anions and Organic Acids in Fermentation Broths 123

Anions & CationsDetermination of Residual Trifluoroacetate in Protein Purification Buffers and Peptide Preparations by Reagent-Free™ Ion Chromatography

160

Anions & Cations Assay for Citrate and Phosphate in Pharmaceutical Formulations Using Ion Chromatography 164

Anions & Cations Direct Determination of Cyanate in a Urea Solution and a Urea-Containing Protein Buffer 200

Anions & CationsDetermination of Inorganic Anion Impurities in a Water-Insoluble Pharmaceutical by Ion Chromatography with Suppressed Conductivity Detection

220

Carbohydrates Quantification of Carbohydrates and Glycols in Pharmaceuticals 117

Carbohydrates The Determination of Carbohydrates, Alcohols and Glycols in Fermentation Broths 122

Get your Free

Pharmaceutical A

pps Notebook

at www.dionex.com/pharmanotebook

Global Pharmaceutical Solutions

Passion. Power. Productivity.

Formidable Challenges on a Global Scale

page 2

The challenges faced by pharmaceutical companies are many. In addition to the increasing financial pressure from competition, unethical suppliers are resorting to product adulteration.

It is imperative to continually look for opportunities to reduce the cost per analysis, increase analytical productivity, and develop greater flexibility.

Liquid Chromatography (LC) is the most common analytical technique used in the pharmaceutical industry. From discovery to manufacturing, LC systems are used both as stand-alone tools and as front-ends for mass spectrometers.

LC separations come in a variety of configurations. While reversed-phase separation is by far the most popular technique, other separation mechanisms are also important. Hydrophilic-interaction liquid chromatography (HILIC), ion-exchange, and mixed-mode separations have found their way into the pharmaceutical chemist’s toolbox.

The pharmaceutical industry faces formidable challenges. Many popular drug patents are expiring. However, developing a new drug may cost approximately 1 billion USD over 10 years, with over 10,000 compounds being tested often yielding only one licensed product. Furthermore, political pressure globally to control health care costs has drawn attention to the high costs of pharmaceutical products. Biopharmaceuticals have exploded on a global scale. International agencies are striving for uniform regulatory control. While all these challenges are mounting, the global marketplace is shrinking. The solution is to bring new pharmaceutical products to the market in a more efficient and economic way.

Dionex understands the demands placed on the pharmaceutical industry and the variety of options available. Our separation and detection technologies, combined with experience and application competence, provide solutions for analyzing a wide range of compound classes. All the top 10 pharmaceutical companies use Dionex equipment for the analysis of small drug molecules, counterions, biologics, oligosaccharides, and many more.

Dionex leads the field in providing solutions to the most complex analytical problems faced by the pharmaceutical industry.

page 3

System Solutions

Liquid ChromatographyThe Dionex UltiMate® 3000 series of HPLC systems are ideal for the analysis of active pharmaceutical ingredients (API), metabolites, and excipients. Choose from a wide variety of configurations to adapt the instrument to your application. From legacy HPLC methods, to high-speed UHPLC separations, to multiplexed tandem or parallel applications, there is an UltiMate 3000 system solution that is right for your lab.

• Excellent retention time precision, detector sensitivity, linearity, and drift performance

• x2 Dual systems for multiplexed tandem or parallel separations

• Rapid Separation LC (RSLC) systems for fast, high-flow UHPLC

• Biocompatible systems for biopharmaceuticals

• Reversed-phase, ion-exchange, mixed-mode, and monolith columns

• Diode array, multi-wavelength, fluorescence, CAD® Charged Aerosol Detector, and MS detectors

• Integrated control and data processing with Chromeleon® Chromatography Data System (CDS) software

Ion ChromatographySince the development of ion chromatography (IC) over 30 years ago, Dionex has pioneered the development of IC systems, media, and applications.

Whether you are running a few samples for process development or have a heavy workload of samples for QC and regulatory compliance, Dionex has the right system to suit your needs.

• Reagent-Free™ systems for reducedeluent preparation

• Dual systems to facilitate complex methods and double throughput

• Suppressed conductivity, pulsed amperometry, UV-vis, and MS detection technology

• Ion-exchange/exclusion, carbohydrate, amino acid, and organic acid analyses

• Concentrator, polishing, and trap columns for on-line sample preparation and trace analyte determination

• System control and data processing with Chromeleon software

Chromatography Data SystemsSoftware is more than just an essential component of a modern chromatography system—it is often the most important factor in how much you get out of that system.

Whether your needs are basic or complex—whether you use instruments from Dionex, or from other manufacturers or both—we have a Chromeleon CDS software solution that’s right for you.

• Chromeleon CDS software's thoughtfully designed user interface and visual cues guide you naturally through your chromatography

• Find out how much time you can save with Chromeleon CDS version 7 Cobra™ Peak Detection algorithm, SmartPeaks™ Integration Assistant, and dynamic interactive data processing

• Satisfy regulatory requirements without sacrificing efficiency using Chromeleon CDS version 7 software's integrated security system, audit trails, and version management tools

• Simplify your analyses using Chromeleon software's innovative eWorkflows

Chromeleon Chromatography Data System

ICS Series IC/RFIC™ SystemsUltiMate Series HPLC/UHPLC Systems

Drug Discovery

Library Maintenance

Libraries also have to be routinely analyzed to verify their content and purity. The productivity provided by the UltiMate 3000 x2 Dual RSLC system enables the analytical chemist to analyze samples at twice the rate of single HPLC systems.

When configured for parallel operation, as shown in the figure below, the system makes use of the autosampler’s free time to inject the same or a different sample into the second column. This results in doubling the analytical capacity.

As shown below, two different analyses can be performed almost simultaneously. After injecting the first sample (water-soluble vitamins), the autosampler injects the next sample into the second column/detector channel (fat-soluble vitamins).

Compound LibrariesCompound libraries are a key source of potential drug candidates and may contain millions of compounds. Large libraries are needed because screening 10,000 compounds may yield only a few with promising biological activity.

Peptide bead-bound synthetic methods facilitate the production of large libraries.

While information gathered from the Human Genome Project promises to significantly change the future of drug discovery, New Molecular Entities (NMEs) discovered using traditional processes still have the greatest chances of success. Compound libraries are screened, hits are developed into leads, and leads are optimized. This process requires a large number of biochemical assays.

Resin and bifunctional linker unit containing protecting groups to allow for alternating binding for peptide-coded libraries.

Two different samples analyzed in parallel, thus doubling throughput.UltiMate x2 Dual system configured for parallel operation with two columns and detectors.

Analysis of pooled single beads can accelerate the identification process.

Subsequent identification of the products is achieved by transferring the beads to a vial, adding cleavage agents, and analyzing the contents by HPLC as shown in the figure below.

O

N

OCH

CH3

3

O

H

O

(CH2 )4

NH

FMOC

NH MOZ

Resin

27331 0

0.1

0.2

0.3

0.4

UV ab

sorb

ance

(214

nm

)

10 15 20 25Minutes 27332

Inject Time

Water-Soluble Vitamins

Fat-Soluble Vitamins

Equilibration Time

Preparation Time for Valve Switching

Exclusive Access Time

Run Time

Waiting for Exclusive Access* Time of Valve Switching

*

*

*

*

27334

Valve

Autosampler

Dual-GradientPump

from Right Pump

from Left PumpColumn 2

Column 1

Detector

Detector

27333

page 4

Combined extracted ion chromatograms from the capillary LC/MS/MS of a pooled, post-dose rat urine sample.

MS/MS spectrum of 5 pg of test compound (MH+ 433).

Lead Optimization

The high attrition rate of compounds during Phase 2 of clinical trials is a major problem for the pharmaceutical industry. To lessen this problem, greater emphasis is being placed on optimizing a promising compound’s PharmacoKinetics (PK) and PharmacoDynamics (PD). It is therefore imperative that a new drug’s PKPD and toxicology be understood as early as possible.

One important aspect of this investigation is accomplished through in vitro and in vivo studies prior to testing in humans.

The metabolism of a drug by the human body plays an important role in its pharmacological actions. Optimizing the metabolic profile is an important step in moving a compound towards further development.

The parent drug and/or its metabolites are often present in the biological fluid at very low concentrations. Hence, reliable, sensitive, and specific analytical techniques are required for their detection and characterization. HPLC coupled to ion-trap mass spectrometry (MS) is a popular tool for the rapid and effective identification of drug metabolites.

Dionex offers a wide range of HPLC, UHPLC, and IC front-ends for a variety of MS, MS/MS, and MSn platforms. With our DCMSLink™

software, Dionex systems can be controlled seamlessly from the most popular MS data platforms, including Xcalibur®, Analyst®, and HyStar™.

The separation shown below combines the extracted ion chromatograms at m/z 398, 412, 414, and 416 collected from the analysis of post-dose rat urine. The MS/MS spectrum of 5 pg of test compound (MH+ 433) is shown in the figure below.

Metabolite I

Metabolite II Metabolite III

25.216.415.1

Parent Drug

17.9

Relat

ive A

bund

ance

6

4

2

01050 15 20

Minutes 27335

5666 91 107 132

160

201

229

254 277 306 341

379

423

5 pgMS/MS

2: MS/MS (433), Time=14.36 min (#468)

60

100

40

80

20

020015010050 300 350250 400

Mass 27336

%

page 5

page 6

FormulationIn optimizing drug adsorption, it is common to pair an API with a suitable anion or cation to form a more aqueous-soluble salt. This typically requires a separate analysis of the API and potential counterions.

For the past 35 years, Dionex has been the innovation leader in ion analysis. Dionex IC columns are used throughout the industry for analyzing counterions and related impurities (as shown below).

Drug Development

Once an NME has been selected for further development, the analytical challenges accelerate. Before entering clinical trials, formulations must be developed, analytical methods created, and synthetic processes optimized. Flexibility and speed of analysis are key requirements.

In addition to conventional reversed-phase columns, Dionex offers mixed-mode columns that combine the selectivity of ion exchange with reversed-phase columns.

Our latest column innovation, the Acclaim® Trinity™ P1 column, combines anion-exchange, cation-exchange, and reversed- phase functionality to enable the separation of an API and associated counterions in a single separation. By eliminating the requirement for separate API and counterion analyses, formulation chemists can be more productive.

A practical application of this new separation technology is shown in the figure below on the right. The chromatogram shows the separation of an API–in this case, naproxen and its sodium counterion.

Both analytes are easily separated with good peak shape in less than 3 min using a simple acetonitrile and ammonium acetate gradient. Normally, this simultaneous analysis of API and its counterion would not be possible using UV detection because the sodium ion does not absorb UV light. The instrumental technology enabling this analysis is the Corona® ultra™ Charged Aerosol Detector. Also, its UHPLC capability allows the analysis to be performed at a higher linear velocity which reduces the analysis time to under 1 min.

Sulfate counterion and anionic impurities separated on an IonPac®AS11-HC column.

Separation of an API, naproxen, and its counterion under HPLC and UHPLC conditions.

Peaks: 1. Unknown — 2. Acetate 0.080 3. Unknown — 4. Chloride 0.025 5. Carbonate — 6. Sulfate n.d. 7. Phosphate 0.23 8. Pyrophosphate 0.035 9. Unknown —

0.4

4.0

1

23

4

5

6

7

8 9

0 10 20 30

µS

27337Minutes

27338

t 0

0.6 mL/min

2.0 mL/min

Peaks: 1. Na+

2. Naproxen

21

12

Minutes

0 2 431

mV

1000

t 0

0

page 7

Method DevelopmentAnalytical methods developed for NMEs are important as they will accompany the potential drug through development and production. Methods must have sufficient sensitivity, selectivity, speed, and robustness, and are thus not easily selected.

With the UltiMate 3000 system, you have a platform that can be configured to automatically search for the optimum separation conditions. The Automated Method Scouting system can run a sequence of analyses using up to 10 different buffers, 3 different organic solvents, and 6 different columns.

Running many experiments generates a large amount of data. However, by using the Chromeleon system's highly versatile charting capability, determination of the separation optima is quick and easy.

When you are ready, all newly developed methods can easily be accelerated to UHPLC-levels without having to upgrade or replace the existing system.

Process DevelopmentDeveloping optimal manufacturing processes requires complete understanding of the reaction kinetics. In order to fully understand the reaction, it is important to make accurate and timely measurements. On-line or at-line HPLC analyses effectively provide this capability.

Dionex is the only partner with experience migrating bench-scale reaction monitoring through pilot-scale development and on to production.

The same analytical systems used in the lab to verify the results of process optimization are used in our Integral systems for at-line or on-line process monitoring.

Final Product

Starting Product

IntermediateCo

ncen

tratio

n

Time 27341

B

Column Compartment

Column 6

Column 5

Column 4

Column 3

Column 2

Column 1

DAD Detector

3

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54

2 6

3

16

54

2

32

5

10

6

9

87

4

1

Pump

C

Autosampler

D

A1

A2

A3

A4

A5

A6

A7

A8

A9

A10

27339

UltiMate 3000 RSLC system configured for automated method scouting.

Chromeleon bubble plot graphically displays results at a glance.

Reaction kinetic data showing reaction progress as a function of time.

The Integral™ system facilitates technology migration from the laboratory to production.

Cleaning ValidationRegulatory agencies require that the equipment used during the manufacturing process be cleaned of residues from the previous batch and any cleaning solutions used in the process. Ion chromatography is a well-established and popular technique for this analysis.

Drug Production

page 8

There is constant pressure in drug manufacturing to keep the production line moving. Dionex solutions ensure reliable performance in the drug manufacturing environment. Our solutions are easy to implement, require a minimal amount of training, and above all, are secure. Dionex system solutions have enabled us to partner with the world’s leading pharmaceutical and biopharmaceutical companies.

The chromatogram below shows the separation of dimethylbenzenesulfonate (DBS) from the other ingredients found in MICRO®, a solution used for cleaning equipment. DBS is ionic and therefore is easily retained on an ion-exchange resin. Since it absorbs UV light, it can be monitored with UV detection. The minimum quantifiable limit is 0.1 mg/L.

Stability Testing Routine testing to ascertain a product’s chemical stability is also important. High sensitivity and selectivity are both required for positive determination.

Dionex HPLC and IC solutions can provide valuable confidence in knowing that degradants and impurities are not present.

The example shown below uses IC to separate bethanecol, a quaternary ammonium compound pharmacologically related to acetylcholine, from degradation product, 2-hydroxypropyltrimethyl ammonium.

Separation of bethanecol and a degradation product on an IonPac CS14 column.

Separation of MICRO cleaning solution on an OmniPac PAX-100 column.

27343

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5

4

Column: OmniPac® PAX-100Eluent: 5 mM Sodium chloride 0.2 mM Sodium hydroxide 32% AcetonitrileFlow Rate: 1 mL/minInj. Volume: 25 mL Detection: UV at 220 nmPeaks: 1–4. Unidentified 5. Dimethylbenzenesulfonate

27344

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Day 1

Day 5

Column: IonPac CG14 and CS14Eluent: 20 mM MSAEluent Source: EG50Temperature: 30 CFlow Rate: 1 mL/minInj. Volume: 25 LDetection: Suppressed conductivity, CAESSample Prep: Prepared in 0.1 N NaOHPeaks: 1. 2-HPTA 1.1 mg/L 2. Bethanechol 9.5

0.20

µS

4.00

0.20

µS

Peaks: 1. 2-HPTA 9.5 mg/L 2. Bethanechol -

Drug Production

page 9

Regulatory CompliancePharmaceutical companies are faced with a wide variety of regulatory requirements from agencies such as the FDA, USP, EMEA, EP, and ASTM. In such an environment, pharmaceutical companies can easily become mired in time-consuming regulatory processes and standards.

Dionex hardware, software, service, and support enable the regulated lab to meet these requirements with minimum effort, stress, and cost.

The Dionex RSLC, HPLC, and IC system solutions are designed to operate dependably over a wide range of applications.

When help is needed, the Dionex service and support organization is there for you with decades of experience. In addition to preventive maintenance and system repair, complete validation services are available to verify that the system’s performance meets specifications.

When it comes to software control, the Chromeleon CDS software is a recognized leader in chromatography data security. A full suite of security features such as electronic signatures, instrument audit trails, and user account management are included.

The latest release, Chromeleon 7, features an innovative capability—eWorkflows. An eWorkflow reduces the steps needed to perform analyses and ensures procedural rules are followed. These are important benefits for the routine analyses found in quality control and compliance monitoring.

Using an eWorkflow, the operator simply selects an instrument, specifies the number of samples to be analyzed, the starting autosampler vial position, and begins the analysis sequence. The software runs the chromatography, processes the data, and produces fully customizable reports.

Chromeleon software can be described in two words—Simply Intelligent.

System suitability tests are easily programmed into a sequence with fully customizable reporting.

1. Choose eWorkflow and relevant instrument.2. Enter number of samples for analysis and starting vial position.

Product Release TestingThe routine analysis of drug products containing multiple analytes in difficult matrices is not new to the pharmaceutical manufacturing environment. Fortunately, Dionex is a recognized leader in innovative separation chemistries.

To avoid performing one analysis for an API and another for its counterion, a specially designed column, such as the Acclaim Mixed-Mode WAX-1, can be used to separate both in a single analysis.

Analytical Research & Development

page 10

Analytical R&D encompasses a wide range of capabilities and responsibilities. Samples may be submitted from all areas in order to obtain information that can make a difference. The demands in AR&D place a premium on versatility, productivity, reliability, and robustness.

The chromatogram below shows the separation of a basic drug substance along with its counterion, found in an over-the-counter medication. The active ingredient is trimipramine, a tricyclic antidepressant, and the counterion maleate.

Using an acetonitrile and phosphate buffer mobile phase, both analytes can be separated with good peak shapes in less than 8 min.

BiopharmaceuticalsThe use of biological processes to produce biopharmaceuticals has exploded in recent years. As in small molecule drug discovery, understanding the processes that create biologics requires accurate analyses that are often challenging.

Dionex offers a wide range of separation chemistries that enables the chemist to accurately monitor levels of amino acids, carbohydrates, peptides, and proteins.

Analysis of the aminoglycoside antibiotic streptomycin A and an impurity on the CarboPac® PA1 column.

Analysis of a mixture of glycols, sugars, and sugar alcohols on the CarboPac PA10 column.

Simultaneous analysis of a basic drug (trimipramine) and its counterion (maleate) on an Acclaim Mixed-Mode WAX-1 column.

27348

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60

–10

nC

Minutes0 40 50 603010

Dihydrostreptomycin

System Suitability

Peak

Streptomycin A

Late-Eluting Impurity

Channel A: WaterChannel B: 250 mM NaOH

80.0% B (200 mM NaOH)

Flow: 0.500 mL/min

28.0% B (70 mM NaOH)

27347

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AU

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112

N

N

OOH

OOH

Column: Acclaim Mixed-Mode WAX-1, 5 µmDimensions: 4.6 × 150 mmMobile phase: 30/70 v/v acetonitrile/ phosphate buffer, pH 6.0 (50 mM overall)Temperature: 30 °CFlow rate: 1 mL/minInj. Volume: 2.5 µLDetection: UV, 220 nmSample: 0.5 mg/mL of trimipramine maleate

Peaks: 1. Trimipramine 2. Maleate

27349Minutes

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120 Column: CarboPac PA10, PA10 guardEluent: 18 mM Sodium hydroxideFlow Rate: 1.5 mL/minInj Volume: 10 µLDetection: Pulsed amperometry, ED40, gold electrodePeaks: 1. Propylene glycol 2. Glycerol 3. Sorbitol 4. Mannitol 5. Maltitol 6. Glucose 7. Sucrose

page 10

Analytical Research & Development

page 11

Natural ProductsNatural products are a promising source of pharmacologically active compounds but present a unique set of challenges. Many applications are highly complex with interfering matrices and unknown analytes. These factors put an emphasis on separation selectivity and detection sensitivity.

F. chrysanthemi, commonly known in China as wild chrysanthemum, is a common medicinal plant. Flavonoids and homo-chlorogenic acids are two compound classes found in F. chrysanthemi and can be used as markers for identification.

27352

O

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OO

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OOHOHO

HOOH

OHChlorogenic acidLinarin

OHHO

HO

HOOH

OHOH

OHHO

OHOH

OH

OH

OH

OHOHOH

Luteolin-7-o-glucosideLuteolin

OH

HOOH

Apigenin

Five compounds used to create a chemical fingerprint for wild chrysanthemum.

Comparison of eight samples of wild chrysanthemum.

Chromatogram of a F. chrysanthemi sample from a commercial retailer.

UV spectra of (A) chlorogenic acid, (B) luteolin-7-o-glucoside, (C) linarin, (D) luteolin, and (E) apigenin.

0 5 10 15 20 25 30 35 40 45 50 55 60 65–200

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page 11

By creating an HPLC fingerprint, verification of authenticity can be established. In this method, an Acclaim C18 column is used to create a fingerprint based on the peaks for chlorogenic acid and four different flavonoids.

By employing diode array detection, UV spectra can be collected and used to confirm the identity of five compounds of interest as shown on the right.

Fingerprinting provides a practical technique for screening samples of Traditional Chinese Medicines (TCM) for possible adulteration or counterfeiting.