Primer

PHARMACEUTICAL IMPURITY

ANALYSIS SOLUTIONS

3

CONTENTS1. PHARMACEUTICAL IMPURITY ANALYSIS –

OVERVIEW AND REGULATORY SITUATION

The Three Major Categories of Pharmaceutical Impurities .....................................................................4

Organic impurities .............................................................................................................................4

Inorganic (elemental) impurities .......................................................................................................5

Residual solvents ...............................................................................................................................5

Selected Publications and Guidelines for the Control of Pharmaceutical Impurities ..............................7

2. ANALYTICAL TECHNOLOGIES FOR IMPURITY PROFILING IN PHARMACEUTICAL DEVELOPMENT

Fourier Transform Infrared Spectroscopy (FTIR) .....................................................................................9

Preparative Liquid Chromatography (LC) ................................................................................................9

Liquid Chromatography and Ultraviolet Spectrometry (LC/UV) ............................................................10

Liquid Chromatography and Mass Spectrometry (LC/MS) ..................................................................11

Capillary Electrophoresis (CE) ................................................................................................................11

Supercritical Fluid Chromatography (SFC) ............................................................................................12

Nuclear Magnetic Resonance Spectroscopy (NMR) ............................................................................13

Inductively-Coupled Plasma Optical Emission Spectroscopy (ICP-OES) and Inductively-Coupled Plasma Mass Spectrometry (ICP-MS) ...................................................................................................13

Gas Chromatography (GC) ....................................................................................................................14

3. A SELECTION OF AGILENT APPLICATION SOLUTIONS FOR THE THREE MAJOR TYPES OF IMPURITIES

Overview ................................................................................................................................................15

3.1 ANALYSIS OF ORGANIC IMPURITIES ...............................................................................16

Achieve precision, linearity, sensitivity, and speed in impurity analysis with the Agilent 1200 Infi nity Series HPLC/UV Solutions ...................................................................................................16

Improve profi ling productivity for the identifi cation of trace-level impurities using Agilent LC/Q-TOF solutions ..........................................................................................................................20

Quantitative analysis of genotoxic impurities in APIs using Agilent LC/QQQ solutions ..................21

Agilent Organic Impurity Profi ling Publications ...............................................................................23

3.2 ANALYSIS OF INORGANIC IMPURITIES ............................................................................24

Determination of elemental impurities in pharmaceutical ingredients according to USP procedures by Agilent ICP-OES and ICP-MS based solutions ........................................................24

Agilent Elemental Impurity Analysis Publications ...........................................................................25

3.3 RESIDUAL SOLVENT ANALYSIS .........................................................................................26

Faster analysis and enhanced sensitivity in residual solvent analysis as per USP <467> procedures using Agilent GC based solutions .................................................................................26

Agilent Residual Solvent Analysis Publications ...............................................................................28

Appendix: Agilent Solutions for Pharmaceutical Impurity Analysis .......................................................29

4

Pharmaceuticals impurities are the unwanted chemicals that remain with active

pharmaceutical ingredients (API) or drug product formulations. The impurities

observed in drug substances may arise during synthesis or may be derived from

sources such as starting materials, intermediates, reagents, solvents, catalysts, and

reaction by-products. During drug product development, impurities may be formed as

a result of the inherent instability of drug substances, may be due to incompatibility

with added excipients, or may appear as the result of interactions with packaging

materials. The amount of various impurities found in drug substances will determine

the ultimate safety of the fi nal pharmaceutical product. Therefore, the identifi cation,

quantitation, qualifi cation, and control of impurities are now a critical part of the

drug development process.

Various regulatory authorities focus on the control of impurities: the International

Conference on Harmonization (ICH), the United States Food and Drug Administration

(USFDA), the European Medicines Agency (EMA), the Canadian Drug and Health

Agency, the Japanese Pharmaceutical and Medical Devices Agency (PMDA), and

the Australian Department of Health and Ageing Therapeutic Goods. In addition, a

number of offi cial compendia, such as the British Pharmacopoeia (BP), the United

States Pharmacopeia (USP), the Japanese Pharmacopoeia (JP), and the European

Pharmacopoeia (EP) are incorporating limits that restrict the impurity levels present in

APIs as well as in drug formulations.

PHARMACEUTICAL IMPURITY ANALYSIS –

OVERVIEW AND REGULATORY SITUATION1

The Three Major Categories

of Pharmaceutical Impurities

According to ICH guidelines, impurities related to drug substances can be classifi ed into

three main categories: organic impurities, inorganic impurities, and residual solvents.

1. Organic impurities

Organic impurities can arise in APIs or drug product formulations during the

manufacturing process or during the storage of drug substances. They may be

known, unknown, volatile, or non-volatile compounds with sources including starting

materials, intermediates, unintended by-products, and degradation products. They

may also arise from racemization, or contamination of one enantiomeric form with

another. In all cases they can result in undesired biological activity.

Recently, genotoxic pharmaceutical impurities, which may potentially increase

cancer risks in patients, have received considerable attention from regulatory

bodies and pharmaceutical manufacturers. In general, genotoxic impurities include

DNA reactive substances that have the potential for direct DNA damage. Potential

genotoxic impurities include process impurities or degradants, present at trace

levels, which are generated during drug manufacturing and storage. As per FDA and

EMA guidelines, potential genotoxic impurities are to be controlled at levels much

lower than typical impurities. The recommended acceptable thresholds for genotoxic

impurities in pharmaceuticals can be found in the guideline documents published

by the USFDA and EMA (See the selected list of key publications provided at the

end of this section). The ICH M7 guidance on genotoxic impurities is currently under

5

preparation with the working title "M7 Assessment and Control of DNA Reactive

(Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk".

2. Inorganic (elemental) impurities

Inorganic impurities can arise from raw materials, synthetic additives, excipients,

and production processes used when manufacturing drug products. Several

potentially toxic elements may be naturally present in the ingredients and these

elements must be measured in all drug products. A further group of ingredients

may be added during production and must be monitored for elemental impurities

once they are known to have been added. Sources of inorganic impurities include

manufacturing process reagents such as ligands, catalysts (e.g., platinum group

elements (PGE)), metals derived from other stages of production (e.g., process

water and stainless steel reactor vessels), charcoal, and elements derived from other

materials used in fi ltration.

The United States Pharmacopeia (USP) is in the process of developing a new

test for inorganic impurities in pharmaceutical products and their ingredients.

The current Heavy Metals Limit Test (USP<231>) is widely acknowledged to be

inadequate in terms of scope, accuracy, sensitivity, and specifi city, and is due to

be replaced with two new general chapters, Limits (USP<232>) and Procedures

for Elemental Impurities (USP<233>), due to be implemented in 2013. In parallel

with the development of USP<232> and USP<233>, the USP is also introducing a

related method <2232> which is specifi c to dietary supplements.

USP<232> defi nes new, lower permitted daily exposure (PDE) limits for a wider

range of inorganic elemental impurities: As, Cd, Hg, Pb, V, Cr, Ni, Mo, Mn, Cu, Pt,

Pd, Ru, Rh, Os, and Ir. A complete list of regulated elements and PDEs can be found

in Agilent publication 5990-9365EN and the references therein. USP<233> further

defi nes the sample preparation and method validation procedures that should be

used for system suitability qualifi cation of any instrumentation used for the analysis of

elemental impurities in pharmaceutical materials. Validation of analytical instruments

that are used for the new USP<232> and USP<233> methods will be performance

based. USP<233> defi nes the analytical and validation procedures that laboratories

must use to ensure that the analysis is specifi c, accurate, and precise.

3. Residual solvents

Residual solvents are the volatile organic chemicals used during the manufacturing

process or generated during drug production. A number of organic solvents used

in synthesis of pharmaceutical products have toxic or environmentally hazardous

properties, and their complete removal can be very diffi cult. In addition, the fi nal

purifi cation step in most pharmaceutical drug substance processes involves a

crystallization step which can lead to the entrapment of a fi nite amount of solvent

which can act as a residual impurity or can cause potential degradation of the drug.

Residual solvent levels are controlled by the ICH, USP, and EP.

Depending on their potential risk to human health, residual solvents are

categorized into three classes with their limits in pharmaceutical products set

by ICH guidelines Q3C. The use of class I solvents, including benzene, carbon

tetrachloride, 1,1-dichloroethane, 1,2-dichloroethylene, and 1,1,1– trichloroethane,

6

should be avoided. Class II solvents, such as methanol, pyridine, toluene,

N,N-dimethylformamide, and acetonitrile have permitted daily exposure limits

(PDEs). A few examples of common organic solvents which are found as volatile

impurities and have their limits set by ICH guidelines are depicted in Table 1. Class

III solvents, such as acetic acid, acetone, isopropyl alcohol, butanol, ethanol, and

ethylacetate should be limited by GMP or other quality-based requirements.

Table 1. ICH limits for a selected list of common organic solvents found as volatile impurities.

Volatile Organic Impurity Limit (ppm) PDE (mg/day)

Acetonitrile 410 4.1

Chloroform 60 0.6

1,4-Dioxane 380 3.8

Methylene chloride 600 6.0

Pyridine 200 2.0

1,1,2-Trichloroethane 80 0.8

USP <467> 2009 General Chapter contains a more comprehensive method for

residual solvent analysis that is similar to the ICH guidelines developed in 1997.

Here, a limit test is prescribed for class 1 and class 2 solvents while class 2C

solvents are usually determined by non headspace methods due to their higher

boiling point. The limits of detection (LOD) recommended for class 3 solvents are

up to 5000 ppm. When the levels of residual solvents exceed USP or ICH limits,

quantitation is required.

NOTE: Regulatory limits for impurities mentioned in this document are given as examples and may not provide the complete information

needed. For complete, current regulatory information and the latest updates, please check the websites of the various regulatory authorities.

7

Selected Publications and Guidelines for the Control of Pharmaceutical Impurities

Key Topics Title

Guidelines for the control of impurities International Conference on Harmonization (ICH) Q3A (R2) Impurities in New Drug Substances,

25 October 2006

ICH Q3B (R2) Impurities in New Drug Substances, 2 June 2006

Specifi c guidelines for the control of genotoxic

impurities

Genotoxic and Carcinogenic Impurities in Drug Substances and Products: Recommended approaches; US

Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and

Research (CDER); Silver Spring, MD, USA, December 2008

EMA/CHMP/SWP/431994/2007 Rev. 3, Questions and answers on the guideline on the limits of genotoxic

impurities, adopted September 23, 2010

Guideline on the Limits of Genotoxic Impurities, CPMP/SWP/5199/02, EMEA/CHMP/QWP/2513442006;

Committee for Medicinal products (CHMP), European Medicines Agency (EMEA); London 28 June 2006

Pharmeuropa, Vol 20, No. 3, July 2008, Potential Genotoxic Impurities and European Pharmacopoeia

monographs on Substances for Human Use

ICH M7 Guideline (in preparation) for control of Mutagenic genotoxic impurities

Guidelines relevant to analytical methods for

the control of genotoxic impurities

ICH Guidance for Industry: Pharmaceutical Development Q8, (R2); US Department of Health and Human

Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER); Aug, 2009,

http://www.fda.gov/RegulatoryInformation/Guidances/ucm128028.htm

ICH Guidelines, Q9: Quality Risk Management Q9; US Department of Health and Human Services. Food

and Drug Administration, Center for Drug Evaluation and Research (CDER): Rockville, MD, Nov, 2005,

http://www.fda.gov/RegulatoryInformation/Guidances/ucm128050.htm

ICH S2A: Specifi c Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals, April 1996

ICH S2B: A Standard Battery for Genotoxicity Testing of Pharmaceuticals, July 1997

ICH S2 (R1): DRAFT Consensus Guideline (Expected to combine and replace ICH S2A and S2B): Guidance on

Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use, March 6, 2008

Guidelines for the control of elemental

impurities

Elemental impurities – Limits (Pharm. Forum, 2011), 37 (3), Chapter <232>

Elemental impurities – Procedures (Pharm. Forum, 2011), 37(3), Chapter <233>

Guidelines for the control of residual solvents ICHQ3C, International Conference on Harmonization, Impurities Guidelines for Residual Solvents. Federal

Register, 62 (247), 1997, 67377

International Conference on Harmonization, ICH Q3C (R3) Impurities: Guideline for Residual solvents,

November 2005

ICH Topic Q3C (R4) Impurities: Guideline for Residual Solvents, European Medicines Agency, 2010

USP Method 467, US. Pharmacopeia, updated June 2007, USP 32 – NF 18

NOTE: This list is a limited selection of key, recent regulatory publications. For complete, current regulatory information and the latest updates, please check the websites of the various regulatory authorities.

8

ANALYTICAL TECHNOLOGIES FOR IMPURITY PROFILING

IN PHARMACEUTICAL DEVELOPMENT2

An impurity profi le is a description of the identifi ed and unidentifi ed impurities present

in a new drug substance (Source: Guidance for Industry, Q3A Impurities in New

Drug Substances). Impurity profi ling processes usually begin with the detection of

impurities, followed by their isolation and characterization. For all three types of

impurities, it is critical to develop a robust method during process development that

can eventually be validated and transferred to QA/QC. Developing reliable methods

for impurities regulated at very low levels, such as genotoxic impurities, adds further

challenges to this process.

To better detect, identify, quantify, and characterize the impurities present in drug

substances and products, pharmaceutical scientists rely on fast analytical tools with

high sensitivity and specifi city. Major analytical tools for impurity analysis include

spectroscopy, chromatography, and various combinations of both, i.e. tandem

techniques. The appropriate technique is selected based on the nature of the

impurity and the level of information required from the analysis. There are various

complex analytical problems in pharmaceutical development that require the use of

more than one analytical technique for their solution. Analytical techniques such as

LC/UV, LC/MS, GC/MS, CE/MS, and LC/UV provide the orthogonal detection and

complementary information that can address these challenges in a time effi cient

manner. As a result, they play a vital role in impurity profi ling of pharmaceuticals from

identifi cation to the fi nal structure elucidation of unknown impurities.

Table 2 summarizes of some of the techniques used in impurity analysis. Further

details on key single and tandem techniques for impurity profi ling are found

in the section that follows.

Table 2. Impurity analysis techniques.

Type of Impurity Technologies

Organic impurities FTIR, Preparative LC, LC/UV, LC/MS (SQ, Q-TOF,

and QQQ), CE, SFC, and NMR

Inorganic/elemental impurities ICP-OES and ICP-MS

Residual solvents GC and GC/MS

See sections below for defi nitions of abbreviations.

Overview

9

Fourier Transform Infrared

Spectroscopy (FTIR)

FTIR is very helpful for identifying and confi rming the structure of an impurity or

degradant because it provides a complex fi ngerprint that is specifi c to a particular

compound. An FTIR spectrum of an organic molecule is determined by the functional

groups present. The technique helps to identify the structure and measure the

concentration of the compound under investigation. Changes in the structure can be

correlated with the help of an FTIR spectrum of a patent drug compared to that of the

impurity or degradant.

Agilent Cary 630 FTIR 

Figure 1. Agilent MicroLab software displays analysis results for the level of ethylene glycol, an impurity

in glycerol. The red color band shows that the level of impurity is outside specifi cation range. See Agilent

publication 5990-7880EN.

The Agilent Cary 630 FTIR packs a powerful combination of precision and compliance,

making it one of the best FTIR systems for routine analysis in pharmaceutical

laboratories. Measuring contaminants, such as ethylene glycol and diethylene glycol

in glycerol, is quick and easy with the 630 FTIR, because its DialPath accessory

reduces the tedious process of fi nding the right path length and optimum measurement

conditions. In addition, Agilent MicroLab software makes it easy to meet regulatory

requirement 21 CFR 11 by alerting users when the impurity level is outside

specifi cation range (Figure 1), while proprietary liquid analysis technology simplifi es

sampling and reduces the risk of user error.

Preparative Liquid

Chromatography (LC)

Since the impurities in the drug substance are usually present at very low quantities,

detailed analysis is only possible upon isolation of the impurities. However, this is a

major challenge in pharmaceutical laboratories. Preparative LC helps isolate impurities

(usually from impurity-enriched analytes, such as the solution remaining from the

crystallization of APIs) in suffi cient quantities to carry out structural analysis, usually

using techniques such as FTIR, NMR, LC/MS, or GC/MS.

Agilent 1260 Infi nity Purifi cation Systems

10

Liquid Chromatography and

Ultraviolet Spectrometry

(LC/UV)

A number of impurity analysis methods found in pharmaceutical quality control

(QC) laboratories use high-performance liquid chromatography (HPLC) coupled

with UV detection (HPLC/UV methods). UV spectrometry helps identify impurity or

degradants in drug substances based on absorption maxima. This technique is one

of the most important and versatile analytical methods available for impurity profi ling

today due to its high selectivity (i.e., ability to quantitatively determine a number of

the individual components present in a sample using a single analytical procedure),

especially for routine analysis where standards are available. Newer, stationary

phase systems are available which operate in several modes, such as ion pairing,

increased hydrophobic interactions, and variable pH, allowing a variety of samples

to be analyzed concurrently based upon their unique properties. High resolution is

particularly helpful when using LC/UV analysis for impurity detection, because all

impurities can be identifi ed with less chance of error. Figure 2 demonstrates the

results achieved using an Agilent LC system combined with Agilent 1.8 μm RRHD

columns identifying and quantifying seven impurities.Agilent 1200 Infi nity Series LC Systems and columns

Figure 2. This data demonstrates the value of UHPLC systems, like the Agilent 1290/1260/1220 Infi nity

Series systems, for impurity analysis. When combined with Agilent 1.8 μm RRHD columns, it was

possible to identify all seven impurities with good baseline separation for accurate quantifi cation. Agilent

Technologies, unpublished data.

Isocratic Impurity Method

Column: 4.6 x 150 mm, 5 µm

4.6 x 150, 5 µm

Rs = 1.15

G/N = 42

4 impurities baseline

not separated for 2

4.6 x 150, 3.5 µm

Rs = 1.37

S/N = 50

7 impurities baseline

not separated for 6

4.6 x 150, 1.8 µm

Rs = 1.80 (+57 %)

S/N = 44

7 impurities baseline

separated for all

mAU

2.5

2

1.5

1

0.5

0

0 5 10 15 20 min

-0.5

-1

11

Liquid Chromatography and

Mass Spectrometry (LC/MS)

LC/MS is a powerful analytical tool that is routinely used in pharmaceutical

development to test and identify product impurities. The detection limit of a few

hundred ppm is readily achievable, ensuring the identifi cation of all the impurities

present at concentrations greater than 0.1 %. MS-based methods generally provide

additional robustness and ruggedness compared to techniques such as UV alone, due

to their high specifi city and sensitivity. While single quadrupole mass spectrometers

work well as analytical tools for the confi rmation of known impurities and the

preliminary structural assessment of unknown impurities, highly sensitive Q-TOF

mass spectrometers provide higher resolution and mass accuracy that enables the

unambiguous identifi cation of unknown trace impurities, making them very useful for

genotoxic impurity analysis. MS-based methods are often selected for the impurity

profi ling of APIs during process development, while UV-based methods are generally

used to test for genotoxic impurities in QC laboratories at manufacturing sites.

Triple-quadrupole (QQQ) LC/MS/MS systems have become a standard platform

for the quantitative analysis of organic impurities in pharmaceutical analytical

laboratories. Combining multiple reaction monitoring (MRM) with a triple

quadrupole tandem mass spectrometer, such as the Agilent 6400 Series QQQ,

enables extraordinary sensitivity for multi-analyte quantitative assays. MRM assays

are particularly useful for the targeted analysis of compounds present in complex

mixtures and matrices, such as blood.

6100 Series Single Quad 6500 Series Q-TOF

6400 Series Triple Quad

Agilent Mass Spectrometers

Capillary Electrophoresis (CE) The determination of drug-related impurities is currently the most important task for

CE within pharmaceutical analysis because it achieves high separation effi ciencies

compared to other chromatographic techniques. CE can be employed when

HPLC techniques are not able to adequately measure impurities, especially in the

case of very polar compounds. A detection limit of 0.1 % is widely accepted as a

minimum requirement for a related impurities determination method and this can be

achieved using CE. In addition, CE is very useful for the separation of closely related

compounds, such as diastereomers and enantiomers. An example of the value of CE

in impurity analysis can be demonstrated using heparin (a polymeric anticoagulant)

as an example. In this case, standard chromatography failed to distinguish drug lots

associated with adverse events while CE was easily able to identify an unknown

impurity (Figure 3). As a result, the use of CE helped to solve this analytical challenge.

Agilent 7100 CE instrument

Figure 3. Capillary electrophoresis of heparin and related impurities using highly concentrated buffers in a

25μm bubble cell capillary. See Agilent publication 5990-3517EN.

2

0

10

20

30

40

50

60

mAU

4 6 8 min

12

Supercritical Fluid

Chromatography (SFC)

SFC, which uses supercritical CO2 as mobile phase, is another orthogonal technique

that can be used for impurity detection because it offers HPLC-level sensitivity with

reduced organic solvent usage (Figure 4). SFC also offers the advantage of chiral

impurity analysis enabling the determination of enantiomeric excess at very low

impurity levels (Figure 5).

Agilent 1260 Infi nity Analytical SFC System

Figure 4. Isocratic separation of the impurity (0.05 % w/w level) from the main component (A) caffeine

and(B) estriol; the signal-to-noise for the impurity at the 0.01 % level is well above 2 – 3, which is usually

the level of detection (LOD). See Agilent publication 5990-6413EN.

min1 2 3 4 5 6 7 8 9

mAU

0

200

400

min1 2 3 4 5 6 7 8 9

mAU

0

200

400

R - 3

S

R

S - 3

R = 1.5

R = 1.7S

R

Figure 5. Determination of enantiomeric excess at impurity levels below 0.05 % using SFC. Chromatograms

of R-1,1’-bi-2-napththol (A) and S-1,1’-bi-2-naphthaol (B) at 5000 ppm. See Agilent publication 5990-5969EN.

mAU

15

10

5

0

-5

1 2 3 4 5 min

mAU

30

20

10

0

-10

1 2 3 4 5 min

Caffeine

Estriol

Caffeine

Estriol

X

X

Mai

n

Mai

n

A

A

B

B

13

Nuclear Magnetic Resonance

Spectroscopy (NMR)

NMR is a powerful analytical tool that enables the study of compounds both in solution

and in the solid state. It has wide applicability because it provides specifi c information

about bonding and stereochemistry within a molecule, which is particularly important

in the structural characterization of drug impurities and degradants often present

only in extremely limited quantities. The non-destructive, non-invasive nature of

NMR spectroscopy makes it a valuable tool for the characterization of impurities and

degradants present at very low levels. NMR can also provide quantitative output, an

important aspect of impurity profi ling.

Agilent 7700 Series ICP-MS

400-MR DD2 Magnetic Resonance System

Inductively-Coupled

Plasma Optical Emission

Spectroscopy (ICP-OES) and

Inductively-Coupled Plasma

Mass Spectrometry (ICP-MS)

The new draft elemental impurities procedure (USP<233>) requires that an

instrument-based method is used to determine elemental impurities and that the

reference methods are based on either ICP-MS or ICP-OES. With both methods,

sample analysis can be accomplished in three ways: directly (unsolvated), following

sample preparation by solubilization in an aqueous or organic solvent, or after acid

digestion using a closed-vessel microwave system.

ICP-OES

ICP-OES provides parts per billion (ppb) detection limits for most regulated elements

in pharmaceutical products, easily meeting the specifi ed limits in cases where direct

sample analysis or small dilution factors are appropriate. It also provides extended

dynamic range, robust plasma, and one-step measurement of major, minor, and trace

elements. Therefore, ICP-OES addresses the needs of a wide range of users, including

those seeking a cost-effective solution for the direct analysis of elemental impurities in

bulk raw materials and pharmaceutical products.

ICP-MS

ICP-MS is a powerful and sensitive technique that delivers a reliable trace-level

analysis of all 16 elements whose limits are defi ned in USP<232>. The low detection

limits of ICP-MS ensure that all regulated elements in drug substances or drug products

can easily be determined using the new method, at or below regulated levels, and even

when large sample dilutions are required. ICP-MS can also be used in combination

with a variety of separation techniques, such as HPLC, GC, and CE, providing several

options for separation (or speciation) of the different chemical forms of the elements,

and depending upon the nature of sample. ICP-MS achieves low detection limits for

almost all elements, including those found in the more extensive analyte list proposed

in the ICH Q3D, such as Au and Tl.

Agilent 720 and 730 ICP-OES

14

7890A/5975C GC/MS system

with 7697A Headspace sampler

GC columns

Gas Chromatography (GC) In combination with fl ame ionization detection (FID), GC is the standard choice

for the analysis of volatile organic impurities, such as residual solvents. The gas

chromatography headspace method is used worldwide for residual solvent analysis

in quality control laboratories because it closely follows ICH Q3C guidelines. Sample

preparation and introduction is via a static headspace which facilitates the selective

introduction of volatile solvents without contamination by mostly non-volatile drug

substance or drug products. Therefore, the use of an FID detector helps preferentially

identify and quantify residual solvents. More recently, the combination of gas

chromatography and mass spectroscopy (GC/MS) has been successfully used for

confi rmation and identifi cation purposes, highlighting the fl exibility of this technology.

15

A SELECTION OF AGILENT APPLICATION SOLUTIONS

FOR THE THREE MAJOR TYPES OF IMPURITIES3

3.1 ANALYSIS OF ORGANIC IMPURITIES

• Achieve precision, linearity, sensitivity, and speed in impurity analysis with the

Agilent 1200 Infi nity Series HPLC/UV solutions

• Improve profi ling productivity for the identifi cation of trace-level impurities

using Agilent LC/Q-TOF solutions

• Quantitative analysis of genotoxic impurities in APIs using Agilent

LC/QQQ solutions

3.2 ANALYSIS OF INORGANIC IMPURITIES

• Determination of elemental impurities in pharmaceutical ingredients according

to USP procedures by Agilent ICP-OES and ICP-MS based solutions

3.3 ANALYSIS OF RESIDUAL SOLVENTS

• Faster analysis and enhanced sensitivity in residual solvent analysis as per

USP <467> procedures using Agilent GC based solutions

This section includes a selection of detailed examples of Agilent applications

solutions that have been developed to meet the challenges encountered when

analyzing the three types of pharmaceutical impurities: the qualitative and quantitative

analysis of trace level organic impurities, the determination of elemental impurities, and

the analysis of residual solvents according to USP procedures.

Overview

16

ANALYSIS OF ORGANIC IMPURITIES3.1

Achieve precision, linearity,

sensitivity, and speed in

impurity analysis with the

Agilent 1200 Infi nity Series LC

Figure 6. Analysis of amoxicillin and fi ve impurities using the Agilent 1220 Infi nity LC System and a gradient

method in combination with UV detection, an Agilent ZORBAX SB-Aq column, and ChemStation software.

See Agilent publication 5990-6093EN.

mAU

8

6

4

2

0

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 min

Impu

rity

A

Impu

rity

C

Impu

rity

B

Impu

rity

D

Impu

rity

E

Am

oxi

cilli

n

Agilent 1200 Infi nity Series LC/UV Systems are an ideal solution for impurity analysis in

pharmaceutical quality control laboratories seeking to achieve the necessary precision,

linearity, sensitivity, and speed required to meet the regulatory standards for impurity

analysis. The example shown in Figure 6 is for the analysis of amoxicillin and its

impurities. This analysis was completed within 7 minutes and detected impurities down

to a level of 0.01 %. Excellent precision of retention times, peak areas, and linearity

was achieved with a correlation coeffi cient of R2 > 0.999 (Figure 7) for fi ve impurities.

0 2 4

Area

2

1.5

1

0.5

0

Amount (ng/μL)

Impurity D

Correlation: 0.99998

0

0

2

4

6

8

10

Area

2.5Amount (ng/μL)

5 7.5

Impurity A

Correlation: 0.99999

0 2.5 5 7.5

Area

3

2.5

2

1.5

1

0.5

0

Amount (ng/μL)

Impurity B

Correlation: 0.99962

0 5 10

Area

2

1.75

1.5

1.25

1

0.75

0.5

0.25

0

Amount (ng/μL)

Impurity C

Correlation: 0.99987

0 2 4 5

Area

5

4

3

2

1

0

Amount (ng/μL)

Impurity E

Correlation: 0.99987

Figure 7. The impurities in amoxicillin are measured with excellent linearity at six concentration levels.

See Agilent publication 5990-6093EN.

17

Agilent’s UHPLC/UV solutions help achieve higher sensitivity, faster sample throughput,

and signifi cant cost savings in impurity profi ling. Since the 1290 Infi nity LC can be

operated at up to 1200 bar pressure, using a very sensitive DAD detector, signifi cantly

faster methods can be developed for profi ling impurities in a highly productive manner.

This leads to a signifi cant reduction in the cost per analysis.

The Agilent Multi-Method solution for LC is ideally suited for testing experimental

conditions, such as determining the ideal combination of stationary and mobile

phases. It makes scouting stationary and mobile phases a simple, automated task,

especially when short run times are used (e.g., a few minutes on an Agilent 1260 or

1290 Infi nity LC).

The Agilent Intelligent System Emulation Technology (ISET) can be used when there is

a need to transfer the fi nal method optimized on UHPLC to standard HPLC equipment

and columns, especially in regulated QA/QC environments. ISET can be used to

execute new or legacy HPLC methods, delivering the same chromatographic results

without the need to change the original method or modify the instrument hardware.

1290 Infinity LC

with ISET

1100 Series LC

1220 Infinity LC 1260 Infinity LC

1200 Series LC

Method Transfer

Other HPLC or

UHPLC System

Figure 8. Agilent’s ISET system can be used to effi ciently transfer methods from a range of

systems to the fi nal QC environment. See Agilent publication 5990-8670EN.

18

The advantage of using ISET’s seamless method transfer for impurity analysis

is demonstrated in Figure 9. After a method was developed for the analysis of

paracetamol and its impurities on the Agilent 1290 Infi nity LC, the ISET tool emulated

the target LC, an Agilent 1100 Series Quaternary LC System, to determine whether the

method that had been developed was suitable for that system. The method was then

transferred to the 1100 Series LC System. The three chromatograms obtained on the

1290 Infi nity LC System, with and without ISET, and those obtained on the 1100 Series

quaternary LC System are compared in Figure 9.

Agilent 1290 Infinity LC Systemwithout emulation

Agilent 1290 Infinity LC System using ISET to emulate the 1100 Series Quaternary LC

Agilent 1100 Series Quaternary LC System

2 4 6 8 10 12 14 16 18

0

5

10

15

20

25

30

Time (min)

mAU

Figure 9. Overlay of chromatograms at 270 nm obtained for paracetamol and its impurities on the

Agilent 1290 Infi nity LC System (blue), the Agilent 1290 Infi nity LC System with ISET (orange), and

on the Agilent 1100 Series Quaternary LC System (black). See Agilent publication 5990-9715EN.

In addition to LC systems, LC columns can signifi cantly impact the results achieved

in organic impurity profi ling. For example, laboratories performing compendia

analysis with conventional, long, 5 µm totally porous LC columns can benefi t from the

increased speed, resolution, and sensitivity that superfi cially porous, Agilent Poroshell

120 columns provide, without having to replace existing instrumentation. Since USP

and EP guidelines allow for method fl exibility in reducing column length and particle

size, transferring methods to shorter 2.7 µm Poroshell 120 columns can save signifi cant

time, while maintaining performance in the separation. This results in higher throughput

and greater productivity with Agilent Poroshell 120 columns than can be achieved with

conventional 5 µm columns (Figure 10).

19

Figure 10. Rapid analysis of cefepime and related impurities on ZORBAX Eclipse Plus (5 μm) and

Poroshell 120 EC-C18 (2.7 μm) columns. See Agilent publication 5990-7492EN.

min0 5 10 15 20 25

mAU

0

10 0.3

65

0.6

06

0.6

77

1.2

31

3.9

17

min0 5 10 15 20 25

mAU

0

10 0.4

82

0.7

98

0.8

90

1.6

17

5.1

86

min0 5 10 15 20 25

mAU

0

10 0.6

60

0.6

90

0.7

15

0.8

44

1.1

88

1.3

27

2.4

06

7.7

48

min0 5 10 15 20 25

mAU

0

10

2.1

82

2.3

11 2

.43

3 2

.63

2 3

.01

8 3

.19

4

4.4

06

4.6

93

9.4

48

24

.72

8

1.0 mL/min

1.0 mL/min

1.5 mL/min

2.0 mL/min4.6 × 75 mm Agilent Poroshell 120 EC-C18

4.6 × 75 mm Agilent Poroshell 120 EC-C18

4.6 × 75 mm Agilent Poroshell 120 EC-C18

4.6 × 250 mm Agilent Eclipse Plus C18 5 µm

Software can also assist in a number of key tasks required for impurity profi ling. For

example, Agilent OpenLAB ELN guides chemists through the complete workfl ow and

documents all data in a central and secure repository that meets regulatory standards.

Agilent OpenLAB Chromatography Data Software (CDS) software also offers built-

in peak purity evaluations (Figure 11) and lets you generate your fi nal impurity

profi le report right from the CDS. By comparing spectra from the upslope, apex, and

downslope, impurities present at less than 0.5 % can be identifi ed. This can and should

be done as a matter of routine to achieve reliable high-quality data. Custom calculation

functionality in this analytical software helps calculate the total level of impurities for

a complete run and includes a PASS/FAIL notifi cation against user-defi nable limits

depending on the toxicity class of the impurities.

5 %

0.5 %

0.1 %

Figure 11. ChemStation peak purity software can be used to determine impurities present at less than 0.5 %,

based on spectral differences. See Agilent publication 5988-8647EN.

20

Improve profi ling productivity

for the identifi cation of trace-

level impurities using Agilent

LC/Q-TOF solutions

If the method is

MS compatible

If the method is not

MS compatible

Result Ex.

m/z: 268.1543

C14

H21

NO4

HPLC Separation

1

2

3

Develop equivalent

MS Compatible LC methodLC/MS analysis using

Agilent 6540 Q-TOF with

full MS scan followed by

auto MS/MS

Find and identify

impurities by MFE

and MFG based on

the accurate mass

MS data

MSC facilitates

the structure

elucidation of the

impurities

Figure 13. Software-assisted workfl ow for impurity identifi cation and profi ling of pharmaceuticals on

the Agilent 1200 Infi nity Series LC combined with an accurate mass Q-TOF, and MassHunter Qualitative

Analysis and MSC software. Agilent publication in development.

The Agilent 6540 Q-TOF delivers sensitive MS and MS/MS analysis of trace level

impurities in drug substances with sub-ppm mass accuracy. The workfl ow shown in

Figure 13 uses advanced MassHunter data analysis features like molecular feature

extraction (MFE) and molecular formula generation (MFG), along with molecular

structure correlator (MSC) software.

The effective use of this novel workfl ow for impurity profi ling is demonstrated by

the rapid identifi cation and structural elucidation of atenolol and eight impurities

(present at > 0.01 % relative to the API’s UV detection area) as shown in Figure 14.

A unique feature of MSC software helps elucidate the structure of impurities in an

effi cient manner. This workfl ow is streamlined to provide high confi dence, accurate

identifi cation and faster structure elucidation compared to conventional impurity

profi ling which requires multiple platforms and spreads analysis over multiple days.

21

Figure 14. Structure elucidation of atenolol and impurity G demonstrating the wide usability of MSC

software to assign structures for each fragment of atenolol (precursor m/z: 267.1703) and impurity G

(precursor m/z: 268.1543). Agilent publication in development.

x103

0

1

2

3

4

5

6

74.0603

190.0856145.0647 267.1703

56.0499 116.106898.0968

178.0856 208.0960

x103

0

1

2

3

4

5

145.064956.050072.0812 191.0698 268.1543

116.107098.0968 226.1062165.0533

Counts vs Mass-to-Charge (m/z)

20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280NH2

OH

NH

CH3

CH3

O

O

OH

OHNH

CH3

CH3

O

O

OH

OH

NH

CH3

H3C

O

O

OH

OH

NH

CH3

H3C

O

O

OH

OH

NH

CH3

H3C

O

O

OH

OH

NH

CH3

H3C

O

O

NH2

OH

NH

CH3

H3C

O

O

NH2

OH

NH

CH3

H3C

O

O

NH2

OH

NH

H3C

H3C

O

O

OH

OH

NH

H3C

H3C

O

O

NH2

OH

NH

CH3

H3C

O

O

Quantitative analysis

of genotoxic impurities

in APIs using Agilent

LC/QQQ solutions

The Agilent 1200 Infi nity Series LC and Agilent 6400 Series Triple Quadrupole (QQQ),

in combination with Agilent columns and MassHunter software, provide a dependable

solution for the quantitative analysis of genotoxic impurities at the lower detection limits

required by current regulations. Variations in organic modifi er, and column stationary

phases and dimensions, can be used to tune the selectivity, peak capacity, and peak

resolution. This generic approach can be applied in early method development or used

for potential genotoxic impurity screening procedures prior to method optimization.

MRM-based quantitation of nine arylamine and aminopyridine potential genotoxic

impurities (PGIs) at trace levels (well below 1 ppm relative to the API) using an

Agilent 1290 Infi nity Series LC coupled to an Agilent 6400 Series QQQ is demonstrated

in Figure 15. Detection limits for these nine PGIs were below 20 ppb (relative to the

API) using MS/MS. Selectivity in the presence of related impurities was assured

through the use of specifi c quantifi ers and qualifi ers for each PGI. All nine PGIs were

well separated within 9 minutes using an Agilent 150 mm ZORBAX Eclipse Plus C18

RRHD column (2.1 mm id, 1.8 μm). Analysis time can be further reduced to 3 minutes

by using a 50 mm Agilent ZORBAX Eclipse Plus C18 RRHD column. One of the PGIs

(2,6-dichloroaniline) was quantifi ed using a diode array detector (DAD) at a detection

level of 100 ppb relative to the API. The recoveries calculated by comparison of a

standard solution of the PGIs provided accuracy levels of 70 %- 130 %, which are

typical limits in pharmaceutical trace analysis procedures (e.g., limit tests).

22

min2 3 4 5 6 7

mAU

0

10

20

30

40

Chlorhexidine, spiked with 1 ppm PGIsDAD 260 nm

0

0

×10-2

×10-2

×10-2

×102

×10-1

×10-1

×10-1

0

0

0

0

0

0

0

1

1

1

1

1

1

1

1

1

Counts (%) vs. Acquisition Time (min)2 3 4 5 6 7

PGI 5, 166.1>130.0 (80.4%)

PGI 9, 122.1>105.1 (98.5%)

PGI 4, 163.1>120.0 (98.3%)

PGI 6, 150.1>108.0 (3.8%, coelution with API)

PGI 3, 136.1>121.0 (101.7%)

PGI 7, 129.1>93.0 (79.1%)

PGI 8, 128.1>93.0 (Present in API, > 20 ppm)

PGI 2, 126.1>111.0 (96.0%)

PGI 1, 119.1>92.0 (89.6%)

2000

1000

1000

2000

500

1000

100

500

250

Counts vs. Acquisition Time (min)0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4

PGI 5, 166.1>130.0

PGI 9, 122.1>105.1

PGI 4, 163.1>120.0

PGI 6, 150.1>108.0

PGI 3, 136.1>121.0

PGI 7, 129.1>93.0

PGI 8, 128.1>93.0

PGI 2, 126.1>111.0

PGI 1, 119.1>92.0

Figure 15. DAD result and quantifi er MRM transitions for the analysis of a chlorohexidine sample spiked with PGIs. A comparison is shown between results achieved

with 150 mm column (A) and 50 mm column (B) Agilent ZORBAX Eclipse Plus C18 RRHD (2.1 mm id, 1.8 μm) columns. Transitions and calculated recoveries are also

indicated. See Agilent publication 5990-5732EN.

A

B

23

Agilent Organic Impurity

Profi ling Publications

Publication Number Title

5990-5732EN Analysis of potential genotoxic arylamine and aminopyridine

impurities in active pharmaceutical ingredients by UHPLC and

UHPLC-MS/MS using the Agilent 1290 Infi nity LC system and the

Agilent 6460A Triple Quadrupole MS system

5990-9715EN Method development on the Agilent 1290 Infi nity LC

using Intelligent System Emulation Technology (ISET) with

subsequent transfer to an Agilent 1100 Series LC - analysis of

an analgesic drug

5990-8670EN Agilent 1290 Infi nity LC with Intelligent System

Emulation Technology

5990-7492EN Fast analysis of cefepime and related impurities

on Poroshell 120 EC-C18

5990-6093EN Analysis of amoxicillin and fi ve impurities

on the Agilent 1220 Infi nity LC system

5991-0115EN Single-run assay and impurity testing of fi xed-dose combination

drugs using the Agilent 1200 Infi nity Series High Dynamic Range

Diode Array Detector Solution

5990-4460EN Quantifi cation of genotoxic "Impurity D" in atenolol by

LC/ESI/MS/MS; with Agilent 1200 Series RRLC and 6410B

Triple Quadrupole LC/MS

5989-7925EN Direct analysis by LC/MS speeds up determination of potential

genotoxins in pharmaceutical drug candidates: AZ success story

5989-5620EN Impurity profi ling with the Agilent 1200 series LC system:

part 4 method validation of a fast LC method

5989-5621EN Impurity profi ling with the Agilent 1200 Series LC System:

part 5 QA/QC application example using a fast LC

5990-3981EN Increasing productivity in the analysis of impurities in

metoclopramide hydrochloride formulations using the Agilent

1290 Infi nity LC System

5990-5819EN Application compendium: analysis of pharmaceuticals and drug

related impurities using Agilent instrumentation

5989-5618EN Isolation of Impurities with Preparative HPLC

5988-8647EN Peak purity analysis in HPLC and CE using diode-array technology

5990-6931EN Highly sensitive UV analysis with the Agilent 1290 Infi nity LC

System for fast and reliable cleaning validation

5990-7880EN Quality verifi cation of incoming liquid raw materials using the

Agilent 5500 DialPath FTIR spectrometer

24

ANALYSIS OF INORGANIC IMPURITIES3.2

Determination of elemental

impurities in pharmaceutical

ingredients according to

USP procedures by Agilent

ICP-OES and ICP-MS

based solutions

In combination with closed-vessel microwave digestion and sample stabilization

using HCl, the Agilent 7700x ICP-MS has been shown to be capable of determining

all regulated elements at low levels in typical pharmaceutical sample digests (See

Agilent publication 5990-9365EN). Simple method development and routine operation

are provided by the standard helium (He) mode method, which uses a single set of

consistent instrument operating conditions for all analytes and samples. As required

in USP<233>, unequivocal identifi cation and verifi cation of analyte results is also

provided by the secondary (qualifi er) isotopes measured in He mode.

Low limits of detection are particularly important for potentially toxic trace elements

defi ned in USP<232>, notably As, Cd, Hg, and Pb. Calibrations for these elements in

He mode are shown in Figure 17, together with Pd and Pt, which are representative

members of the platinum group elements (PGEs) that must be monitored when added

as catalysts as per USP<232>.

Figure 16. The robust plasma system of the Agilent 700 Series ICP-OES ensures the stable analysis of diffi cult

samples, such as the 5 % NaCl brine solution shown here. Agilent Technologies, unpublished results.

The new methodology for the preparation and analysis of pharmaceutical samples

described in the draft General Chapters USP<232> and <233> provides an

opportunity for pharmaceutical laboratories to update their methodology and

instrumentation to address the limitations of the current heavy metals limit test

(USP<231>). The robust plasma system on the Agilent 700 Series ICP-OES is capable

of analyzing the most challenging samples, such as undiluted organic solvents and

concentrated salt solutions, to enable fast, accurate analysis which is free of complex

sample digestion procedures (See Figure 16).

6

4

2

00 30 60 90 120 150 180 210

PP

M

Time (min)

As 188.980

Cr 267.716

Ba 455.403

Mn 257.610

Co 238.892

Se 196.026

Sr 407.771

Zn 213.857

25

Figure 17. Calibration curves for As, Cd, Hg, Pb, Pd, and Pt in He mode, demonstrating limits of detection of

1 ng/L or below, and good sensitivity and linearity for all elements including Hg, Pd, and Pt, which require

stabilization in HCl. See Agilent publication 5990-9365EN.

x10–1

1

0.5

05.0 10.0 15.0

Rat

io

Conc (ppb)

x10–3

2

02.0 4.0

Rat

ioConc (ppb)

As Cd

x10–2

1

0.5

1.5

05.0 10.0 15.0

Rat

io

Conc (ppb)

Hg

x10–1

1

0.5

0

5.0 10.0

Rat

io

Conc (ppb)

x10–1

2

1

3

0

5.0 10.0

Rat

io

Conc (ppb)

Pb Pd

x10–1

5

0

5.0 10.0R

atio

Conc (ppb)

Pt

75 As [He] ISTD: 45 Sc [He] 111 Cd [He] ISTD: 159 Tb [He] 201 Hg [He] ISTD: 209 Bi [He]

208 Pb [He] ISTD: 209 Bi [He] 105 Pd [He] ISTD: 159 Tb [He] 195 Pt [He] ISTD: 209 Bi [He]

R = 0.9998 R = 0.9999 R = 0.9999

R = 0.9999 R = 0.9999 R = 0.9999

System performance validation of the 7700x ICP-MS delivered data that was easily

within method requirements for accuracy, stability, and spike recovery at detection

limits that were all several orders of magnitude lower than the levels at which the trace

elements are currently controlled. This provides the reassurance that the Agilent 7700x

will be able to meet the regulatory requirements for pharmaceutical materials regulated

under USP methods, even if control limits are made more sensitive in the future.

The Agilent 7700x also provides a full mass spectrum screening capability, is tolerant

of all commonly-used organic solvents, and can be linked to a chromatography

system to provide integrated separation and analysis of the different forms of As and

Hg, as required under USP<232>.

Agilent Elemental Impurity

Analysis Publications

Publication Number Title

5990-5427EN Pharmaceutical analysis by ICP-MS: new USP test for

elemental impurities to provide better indication of

potentially toxic contaminants

5990-9365EN Validating the Agilent 7700x ICP-MS for the determination

of elemental impurities in pharmaceutical ingredients

according to draft USP general chapters <232>/<233>

5990-9382EN Proposed new USP general chapters <232> and <233>

for elemental impurities: The application of ICP-MS for

pharmaceutical analysis

5990-9073EN Regulatory compliance for ICP-MS

26

RESIDUAL SOLVENT ANALYSIS3.3

Faster analysis and enhanced

sensitivity in residual solvent

analysis as per USP <467>

procedures using Agilent

GC-based solutions

Quality assurance laboratories routinely use United States Pharmacopeia (USP) method

<467> for residual solvent analysis. The Agilent 7697A Headspace Sampler coupled

to an Agilent 7890 GC offers a very effi cient solution for the analysis of UPS<467>

class 1 and class 2 residual solvents at their limit concentrations in aqueous solutions.

USP<467> specifi es three procedures for class 1 and class 2 residual solvents:

1. Procedure A: identifi cation and limit test

2. Procedure B: confi rmatory test (if solvent is above limit)

3. Procedure C: quantitative test

Procedure A uses G43 phase Agilent 624 columns (VF-624ms or DB-624) and

Procedure B uses a G16 phase (HP-INNOWax) column. In general, analytes that

co-elute in one of these phases do not co-elute in the other.

As demonstrated in Figures 18 and 19, the Agilent 7697A Headspace sampler

is capable of outstanding repeatability for the analysis of residual solvents.

Repeatability is better than 2.5 % relative standard deviation (RSD) for class 1,

class 2A, and class 2B solvents.

An inert sample path, thermal zones with set point stability of better than

+/- 0.1 °C, and EPC-controlled vial sampling using absolute pressure,

all contribute to system performance. Carryover is essentially non-existent in all

confi gurations. User programmable fl ow rates and times, needle/loop purges,

and vent line purges are effectively used to clean the system between runs.

Laboratories should perform system suitability studies and validate their proposed

methods according to USP or ICH guidelines.

For new drug development and quality control, a dual-channel confi guration using

both FID and a mass selective detector (MSD) is a powerful tool for residual solvent

determinations, especially when unknown identifi cation or confi rmation is needed.

This system is particularly well-suited for the development of generic methods

that do not need to follow USP<467> guidelines. MSD analysis also helps avoid

ambiguity, as over 60 solvents are currently used in pharmaceutical manufacturing.

When unknown peaks or solvents are present, this system may be the best

solution for confi rmation and quantitation.

27

1. methanol2. acetonitrile3. dichloromethane4. Trans-1,2-dichloroethene5. Cis-1,2-dichloroethene6. tetrahydrofuran7. cyclohexane8. methylcyclohexane9. 1,4-dioxane10. toluene11. chlorobenzene12. ethylbenzene13. m-xylene, p-xylene14. o-xylene

B

1 2

3

4

5

6

7

8

9

10

11

12

13

14

1. hexane2. nitromethane3. chloroform4. 1,2-dimethyoxyethane5. trichloroethene6. pyridine7. 2-hexanone8. tetralin

1

2

3

4

5

6

7 8C

Figure 18. Class 1 (A), class 2A (B), and class 2B (C) solvents at USP<467> limit concentrations.

See Agilent publication 5990-7625EN.

Figure 19. Class 2A solvents at limit concentrations with FID-MSD. See Agilent publication 5990-7625EN.

TIC

FID

1. 1,1-dichlorothene2. 1,1,1-trichloroethane3. carbon tetrachloride4. benzene5. 1,2-dichloroethane

A

12

3

4

5

28

Agilent Residual Solvent

Analysis Publications Publication Number Title

5990-7625EN Analysis of USP <467> residual solvents with improved

repeatability using the Agilent 7697A Headspace Sampler

5989-8085EN Simultaneous dual capillary column headspace GC with

fl ame ionization confi rmation and quantifi cation according

to USP <467>

5989-9726EN A generic method for the analysis of residual solvents in

Pharmaceuticals using static headspace GC-FID/MS

5990-5094EN Fast analysis of USP <467> residual solvents using the

Agilent 7890A and low thermal mass (LTM) system

5989-6079EN Improved retention time, area repeatability and sensitivity

for analysis of residual solvents

5989-3196EN The determination of residual solvents in pharmaceuticals

using the Agilent G1888 headspace/6890N GC/5975

inert MSD system

29

Appendix: Agilent Solutions for Pharmaceutical Impurity Analysis

Agilent leads the industry with a wide range of instrumentation, LC and GC column choices, and software and informatics solutions

for impurity analysis.

Instrumentation Category of Impurity Application Agilent Instrumentation

Organic impurities Impurity detection and rapid method scouting/development 1200 Infi nity Series LC + Diode-array Detector SL

Detection of impurities not easily separated by HPLC (e.g.,

highly polar compounds)

7100 CE System

Detection of chiral impurities 1260 Infi nity Analytical SFC System

Isolation of impurities 1260 Infi nity Preparative-scale Purifi cation System

Identifi cation of impurity structure 600-IR series FTIR + 400-MR DD2 Magnetic Resonance System +

1200 Infi nity Series LC + 6100 Series Single Quadrupole or 6200 Series

Accurate-Mass TOF or 6500 Series Accurate-Mass Q-TOF LC/MS

Systems (for trace level genotoxic impurities)

Quantitation of genotoxic impurities 1200 Infi nity Series LC + 6400 Series Triple Quadrupole LC/MS Systems

Inorganic impurities Analysis of elemental impurities in pharmaceutical

ingredients - basic requirements of USP that do not

necessitate the lowest detection limits

700 Series ICP-OES

Analysis of all 16 regulated elements at and below the

regulated levels in the new USP <233> method, even when

large sample dilutions are required

7700 Series ICP-MS

Speciation of certain regulated elements (As and Hg) 1200 Infi nity Series LC + 7700 Series ICP-MS

Residual solvents Analysis per USP <467> procedures 7890A GC + 7967A Headspace sampler

Analysis involving unknown peaks/solvents 7890A GC + 5975C GC/MS system + 7697A Headspace sampler

Columns and Supplies

Agilent offers a comprehensive portfolio of GC and LC columns, and supplies for chromatography, spectrometry, and spectroscopy, all

meeting ISO 9001 standards to ensure maximum instrument performance and reproducible results.

Agilent leads the LC industry with column choices that meet a wide range of analytical needs and support the pharmaceutical lifecycle

with maximum scalability across laboratory development settings, and around the world service and support. For example Poroshell

120 columns can save signifi cant analysis time, and Rapid Resolution High Defi nition (RRHD) columns offer maximum fl exibility in

solvent selection and fl ow rates. Agilent also has the broadest portfolio of GC columns available, including innovative options like our

ultra inert GC columns.

Agilent’s comprehensive portfolio of supplies including vials, syringes, gas management, fl ow meters, leak detectors, fi ttings, tools, and

standards, all engineered or selected by our instrument design teams, manufactured to our demanding specifi cations, and tested under

a variety of conditions.

Software and Informatics

Agilent’s industry leading software and informatics portfolio is continuously expanding to cover a broader range of analytical

workstations, data and laboratory management solutions, and applications to satisfy the growing needs of the life sciences

and chemical industries. Agilent software solutions are integrated to address the complete life cycle of scientifi c data, including

experimental design, data acquisition, knowledge management, and analysis in an open system architecture. The Agilent OpenLAB

Software Suite includes OpenLAB Chromatography Data System (CDS), OpenLAB Enterprise Content Manager (ECM), and OpenLab

Electronic Lab Notebook (ELN).

Laboratory Qualifi cation and Testing Solutions

You can count on Agilent to provide the system qualifi cation services or proof of calibration that you need

to support your GLP/GMP or ISO/IEC 17025 quality initiatives. Agilent has been ranked #1 in compliance

since 1995. With the delivery of over 100,000 successful instrument qualifi cations and over a decade of

practical experience in quality testing, you can trust Agilent to deliver confi dence in your analytical results.

30

Learn more

www.agilent.com/lifescience/pharma

Find a local Agilent customer center

www.agilent.com/chem/contactus

USA and Canada

1-800-227-9770

agilent_inquiries@agilent.com

Europe

info_agilent@agilent.com

Asia Pacifi c

inquiry_lsca@agilent.com

For Research Use Only. Not for use in diagnostic procedures.

This information is subject to change without notice.

© Agilent Technologies, Inc. 2012

Printed in the USA, April 19, 2012

5991-0090EN