Getting Ready For USP 232, 233, and 2232 - Book

J.A. Nóbrega - C.
Pirola
GETTING READY FOR

USP 232, 233 AND 2232
Microwave-Assisted Sample Preparation
and Determination of Elemental Impurities
in Pharmaceutical Products
Milestone Press
J.A. Nóbrega - C. Pirola
GETTING READY FOR

USP 232, 233 AND 2232
Getting READY for USP 232, 233 and 2232 Layout:
Microwave-Assisted Sample Preparation A. Fenili
in Pharmaceutical Products Cover and Graphics:
A. Fenili
Authors: S. Lorenzi
J.A. Nóbrega
C. Pirola Printed in Italy by:
Ikonos Srl
ISBN 978-88-96006-30-6
June 2017
© 2017 Milestone Srl 1st Edition
All rights reserved under international copyright
conventions. No part of this book may be reproduced
or utilized in any form or by any means, electronic or
mechanical, including photocopyng, recording, or in
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Milestone Press
CONTENTS
INTRODUCTION
The analysts 7
CHAPTER 1
Overview about USP <232>, <233>, and <2232> 13
1. I am used to working with USP <231> and I know it has its
limitations. Could you please remind me its critical points? 15
2. How would you summarize USP <232>? 16
4. Could you please also inform me on what the ICH Q3D is? 18
5. How are elemental impurities classified in the ICH Q3D? 19
7. How can I prepare my laboratory in order to implement
these norms? 20
8. What are the main difficulties that I should expect? 21
9. Which are the key concepts that must be learned? 22
10. How long will the overall implementation in my lab take? 23
CHAPTER 2
Microwave-assisted sample preparation 25
1. USP <232> recommends the use of a closed vessel for
sample digestion. Which are the advantages and difficulties
of using closed vessels? 27
2. Which are the reagents that I should consider for sample
digestion? 28
3. Which are the hazards of these reagents and which are
the main safety issues? 29
4. Do you recommend the use of microwave-assisted
digestion? Why? 30
5. Which are the microwave technologies available?
Which are the pluses and minuses of each? 32
6. Is there any general procedure that I could adopt? 41
7. Which are the steps that I should follow in order to
establish an analytical procedure for sample digestion? 42
8. How could I improve analytical blanks? 43
9. How could I evaluate the efficiency of a digestion
process? 47
10. Which are the main specifications and parameters to
consider when selecting the proper digestion system?
Sample, type, amount, vessel volume? 48
11. What is the role of T and P in a digestion process? 49
CHAPTER 3
Determination of elemental impurities using ICP OES 51
1. What do you mean by ICP OES? 53
2. What is argon plasma? 54
3. How is argon plasma generated? 55
4. What do I measure in ICP OES? 56
5. How do I choose emission lines? 57
6. Which are the components of an ICP OES? 58
7. Which kinds of samples can I introduce in ICP OES? 59
8. How do I operate an ICP OES? Which are the critical
parameters? 60
9. How do you choose between measurements in axial or
in radial position? 62
10. How do I know if an ICP OES is in good operational
conditions? 63
11. How do I calibrate an ICP OES? 64
12. Which kinds of interferences may I expect? 65
13. How do I solve these interferences? 66
14. What is an internal standard? 68
15. What should I expect when applying ICP OES to USP
<232>, <233> and <2232>? 69
CHAPTER 4
Determination of elemental impurities using ICP-MS 71
1. What do you mean by ICP-MS? 73
2. Is this argon plasma the same one used in ICP OES? 74
3. What do I measure in ICP-MS? 74
4. How do I choose isotopes? 75
5. Which are the components of an ICP-MS? 76
6. Which kinds of samples can I introduce in ICP-MS? 77
7. Which are the critical parameters when operating an
ICP-MS? 78
8. How do I know if an ICP-MS is in good operational
conditions? 79
9. How do I calibrate an ICP-MS? 80
10. Which kinds of interferences may I expect? How do
I solve them? 80
11. What is an internal standard? 82
12. What should I expect when applying ICP-MS for USP
<232> and USP <233>? 82
CHAPTER 5
Validation Procedure 85
1. How could I validate developed analytical procedures? 87
2. How could I evaluate accuracy? 87
3. How could I evaluate precision? 88
4. How could I evaluate sensitivity? 89
5. How could I evaluate specificity? 89
6. How could I evaluate sample throughput? 90
CHAPTER 6
Final Remarks 91
APPENDIX A
References 95
AUTHORS
About the Authors 99
INTRODUCTION
THE ANALYSTS
7
The analysts
ANALYST 1 ANALYST 2
(BEGINNER) (CONSULTANT)
9
Introduction
“Work may be hard sometimes. I

have a long experience in carrying
out organic analyses using gas and
liquid chromatography. I know how to
prepare samples and how to determine
several organic compounds, but now I
have a completely different challenge.
I must learn how to establish analytical
procedures in order to determine
metal and non-metal impurities in
pharmaceutical products and in dietary
supplements”.
“Trust me. It is not as hard as you think”.
10 GETTING READY FOR USP 232, 233, AND USP 2232
“Are you kidding? I know I must follow
USP <232>, <233>, and <2232>, but I
am lost. I know I am supposed to use ICP
OES and ICP-MS, but I am not familiar
with these instrumental techniques and
I do not know how to prepare samples,
how to calibrate them and which kinds of
solutions I can introduce in these ICPs”.
“Let’s make a plan. Organize your

questions and we will discuss them step-
by-step, starting from the USP chapters
and proceeding with sample preparation
and ICP measurements. I deliberately
will not provide comprehensive answers;
instead, I will try to give you practical
answers that may help you perform the
required USP determinations using ICP
OES and ICP-MS”.
11
CHAPTER 1
OVERVIEW OF USP <232>, <233>

AND <2232>
13
Overview of USP <232>, <233>
AND <2232>
1. I am used to working with USP <231> and

I know it has its limitations. Could you please
remind me its critical points?
Figure 1. Typical car in the early 1900s
As you know, USP <231> - Heavy Metals was issued

in 1905 and certainly does not reflect decades of
advances in instrumental analysis. According to the
USP’s description, “this test is provided to demonstrate
that the content of metallic impurities, that are colored by sulfide ion
under specified test conditions, does not exceed the Heavy Metals
15
Chapter 1 - Overview of USP <232>, <233> AND <2232>
limit specified in individual monograph in percentage (by weight)

of lead of the test substance, as determined by concomitant visual
comparison with a control prepared from a standard lead solution
(Note: substances that typically will respond to this test are lead,
mercury, bismuth, arsenic, antimony, tin, cadmium, silver, copper
and molybdenum)”. This test, by visual comparison, may be seen as
qualitative and it is used to demonstrate that the metallic impurities
colored by sulfide ions do not exceed a limit of 10 mg/kg. Of course,
kinetic aspects affect the precipitation processes and the visual
observation varies according to the analyst’s interpretation. It may be
said that the determination of 10 elements does not represent the
analytical demand, for consumer safety, of the complexity of medicines
produced nowadays, considering worldwide producers and synthetic
processes. Summarizing, USP <231> is too simple and old-fashioned
for dealing with with the complexity of pharmaceutical products in the
XXI century. USP <231> does not apply to products intended only for
veterinary use, parenteral solutions and dialysates.
2. How would you summarize USP <232>?
USP <232> Elemental Impurities – Limits specifies the

limits of elemental impurities in drug products. According
to the USP, “elemental impurities include catalysts and
environmental contaminants that may be present in
drug substances, excipients or drug products. These impurities may
occur naturally, be added intentionally, or be introduced inadvertently
(e.g. by interaction with processing equipment and the container
closure system)”. So far, a total of 15 elements are specified, but due
to the ubiquitous nature of arsenic, cadmium, lead and mercury, they
must be considered in the risk assessment. The limits of arsenic and
mercury are based upon inorganic forms, but chemical speciation is
required only if the total concentrations exceed the limit values.
USP <233> - Elemental Impurities - Procedures

describes two analytical procedures for the quantitative
determination of elemental impurities. The chapter
also describes the criteria for acceptable alternative
procedures. Procedure 1 can be used for elemental impurities
generally amenable to detection by inductively coupled plasma
optical emission spectrometry (ICP OES). Procedure 2 can be used
for elemental impurities generally amenable to detection by inductively
coupled plasma mass spectrometry (ICP-MS). A general procedure
for closed vessel digestion is also described.
17
4. Could you please also inform me on what the

ICH Q3D is?
Good point. ICH stands for International Conference

on Harmonization. USP is working closely with ICH to
ensure the alignment of its new standards for elemental
impurities with the ICH Q3D Guideline for Elemental
Impurities. The Q3D Guideline is observed by the European Medicines
Agency (EMA). The ICH Q3D Guideline Step 4 was announced on
December, 16, 2014. This guideline applies to new finished drug
products and to new products containing existing drug substances.
It is highly recommended to read this document in order to check its
scope and the products not affected by Q3D. The guideline contains
three parts: (1) the evaluation of the toxicity data for potential element
impurities; (2) the establishment of a Permitted Daily Exposure (PDE)
for each element of toxicological concern; and (3) the application
of a risk based approach to control the elemental impurities in drug
products.
5. How are elemental impurities classified in the
ICH Q3D?
The elements included in the Q3D Guideline were

distributed in three classes based on their toxicity (PDE)
and likelihood of occurrence in the drug product.
-- Class 1: As, Cd, Hg, and Pb (the so-called “Big Four”). These
four elements require an evaluation during the risk assessment,
across all potential sources of elemental impurities and routes of
administration.
-- Class 2: Based on their relative likelihood of occurrence in the drug
product, elements in Class 2 are further divided in subclasses 2A
and 2B.
-- Class 2A: Co, Ni, and V. These elements have a relatively high
probability of occurrence in the drug product and this requires
assessment across all potential sources of elemental impurities
and routes of contamination.
-- Class 2B: Ag, Au, Ir, Os, Pd, Pt, Rh, Ru, Se, and Tl. These
elements have a reduced probability of occurrence in the drug
product.
-- Class 3: Ba, Cr, Cu, Li, Mo, Sb, and Sn. These elements have
relatively low toxicities in the oral route of administration, but may
require consideration of the risk assessment in inhalation and
parenteral routes.
-- Other elements: Al, B, Ca, Fe, K, Mg, Mn, Na, W, and Zn. The
PDEs for these elements were not established due to their low
19
inherent toxicity. This information was directly obtained from the

cited document and, again, it is highly recommended to carefully
read it. These comments were directly obtained in the cited
document and again, please, a careful reading is recommended.
USP <2232> deals with the determination of elemental

contaminants in dietary supplements.
7. How can I prepare my laboratory in order to

implement these norms?
Laboratories of pharmaceutical industries are proficient

in the determination of organic constituents using
chromatographic techniques. The implementation
of USP <232> and USP <233> requires that these
laboratories must be prepared to deal with inorganic elemental
analyses. There are two critical steps that the laboratory infrastructure
and the team must take into account: (1) sample preparation and
(2) multielemental determination using either ICP OES or ICP-MS.
USP <233> recommends a closed vessel digestion and, certainly,
microwave-assisted heating allows the implementation of fast
and effective procedures for sample digestion. On the other hand,
ICPs will require a supply of argon gas (just to give you an idea, the
consumption is around 1 m3 of Ar per hour). The typical reagents
needed are concentrated solutions of pure nitric acid, hydrochloric
acid, hydrogen peroxide, and stock solutions containing the target
analytes. Remember that, as described in Chapter 2, Q8, concentrated
acids are easily purified by sub-boiling distillation.
8. What are the main difficulties that I should

expect?
ICPs are certainly ready for the determination of

analytes required by USP <232> and USP <233>. Lack
of sensitivity may occur for some specific elements in
ICP OES, but this should not be critical for most of
them and literature is demonstrating the positive performance of this
instrumental technique in carrying out this analytical task. Memory
effects may be critical for specific elements, such as Hg and Os,
and a solution medium should be carefully evaluated. A plethora of
analytical applications, developed using both ICP OES and ICP-MS,
demonstrated their capabilities of solving interferences and some
common processes, discussed in Chapter 3, Q12 and Q13, and in
21
Chapter 4, Q10. Sample preparation is usually the most critical step

in inorganic elemental analysis and you must be prepared to learn the
microwave-assisted procedures using closed vessels and the careful
choice of reagents and heating program. Always remember that, in
most situations, you will be working with closed vessels that contain
concentrated acids at a relatively high pressure, of around 20-40 bar,
and at a relatively high temperature, of around 180-240 °C. Be careful!
9. Which are the key concepts that must be

learned?
I would say that the inorganic elemental analysis,

according to USP <232> and USP <233>, will require
the development of know-hows in the multielemental
analysis using ICPs and microwave-assisted sample
preparation. In this context, trace analysis will play a major role and
analysts must be trained on the typical contamination processes and
losses of target analytes. Fortunately, there is a consistent scientific
literature and companies in the instrumentation area have a huge
experience in both areas. Networks with major instrumentation
companies and academic laboratories will certainly speed up the
implementation of these norms in pharmaceutical laboratories.
10. How long will the overall implementation in
my lab take?
It is difficult to discuss a standard timetable that will vary

according to different factors, such as the lab experience,
the budget, and human resources. It is relatively easy to
prepare the required infrastructure, but lab teams must
be trained and knowledge on trace inorganic analysis
must be developed.
23
CHAPTER 2
MICROWAVE-ASSISTED SAMPLE
PREPARATION
25
Microwave-assisted sample preparation
1. USP <232> recommends the use of a

closed vessel for sample digestion. Which are
the advantages and difficulties of using
closed vessels?
Sample preparation in closed vessels presents several

advantages: (1) the loss of analytes by volatilization
is avoided; (2) contamination is controlled because
chemical processes are carried out in a closed
environment; (3) the required volumes of pure and concentrated
reagents decrease; (4) the pressure increase inside the closed vessel,
caused by the generation of gases, increases the boiling point of the
reagents and, consequently, chemical reactions are carried out in more
severe conditions. All these effects lead to a more efficient digestion.
However, the critical aspect is related to the sample mass that we
can work with in a closed vessel. When a sample is rich in organic
components, we will work with a sample mass of maximum 500 mg
in most closed vessels. This limit is less strict for inorganic samples
because lower amounts of gases are generated during the digestion
and, in this case, we can work with sample masses of around 1000
mg. As it will be discussed in Chapter 2, Q4, the use of microwave-
assisted heating enhances a more gradual increase of pressure in a
closed vessel and this aspect is advantageous for safety reasons.
27
Chapter 2 - Microwave-assisted sample preparation
2. Which are the reagents that I should consider

for sample digestion?
Sample digestion should be carried out using reagents

compatible with further ICP OES and ICP-MS
measurements. This means that we must think about
the chemical and physical properties of each reagent
and also on how the concentration of this reagent will affect the
sample introduction by pneumatic nebulization and the processes in
argon plasma, common in both ICP OES and ICP-MS. Taking into
account these aspects, the use of nitric acid and hydrogen peroxide
is highly recommended. On the other hand, the use of phosphoric
and sulfuric acids should be avoided. The use of hydrofluoric acid,
despite its efficient action on samples containing silicon dioxide,
should also be avoided. Finally, there is no need to use perchloric
acid when working with closed vessels. Perchloric acid is extremely
prone to explosions when in contact with organic compounds and
some of its salts, such as potassium perchlorate, are also explosive.
Remember that the oxidant power of nitric acid increases when its
boiling point is increased; this is due to the medium to high pressure
typically developed during digestions in closed vessels.
3. Which are the hazards of these reagents and
which are the main safety issues?
Working with concentrated acids is potentially critical

and analysts must read their Material Safety Data
Sheets (MSDS) and pay attention to potential health
effects and first aid measures. Please pay attention to
this. Certain reagents are especially critical and I recommend to pay
special attention to perchloric and hydrofluoric acids that preferentially
must not be used. Additionally, remember that you are promoting
chemical reactions in closed vessels and these vessels must have
mechanical, thermal, and chemical resistance. Be careful about the
sample mass and reagent volumes, and reduce both amounts if
possible. Remember that heating acids, and even water, in closed
vessels will generate gases; digestion reactions will also generate
80
70
60
Pressure (bar)
50
40 1.25 g
1.00 g
30
0.75 g
20
0.50 g
10 0.25 g
0 HNO3 only
0 5 10 15 20
Time (minutes)
Figure 2. Organic sample mass vs developed pressure
29
gases (Figure 2). In other words, both processes will increase the
pressure inside the vessel and, consequently, boiling points will also
increase. Vessel materials may be softened at high temperatures
and most polymers used nowadays to build vessels are composed
of modified polyethylene in order to improve thermal resistance and
decrease porosity. These materials resist temperatures of up to 260
o
C in long heating cycles, but a temperature of up to 280 oC may be
used in short heating cycles. However, reaching such temperatures
should be avoided in order not to reduce the vessel’s lifetime. Do not
forget that the boiling point of sulfuric acid, even at ambient pressure
(ca. 335 oC), is higher than the softening point of the vessel material.
It is important to measure both temperature and pressure during the
digestion process. Modern microwave-heated digestion vessels have
proper mechanisms to release pressure. Milestone’s vessels have
the “vent-and reseal” technology that allows us to proceed with the
digestion even after pressure release.
4. Do you recommend the use of microwave-

assisted digestion? Why?
USP <233> recommends the use of a closed vessel

for sample digestion, but the use of the microwave-
assisted heating method to carry out digestions is not
mandatory. I do recommend the use of microwave-
assisted digestions and I would like to point out the following
advantages when compared to conventional heating: (1) microwave
energy is directly absorbed by solutions containing ions and dipoles.
The vessel polymer material is transparent to microwave radiation. It
means that microwave energy is absorbed directly by the medium we
want to heat; (2) as we know from daily life in our kitchens, microwave
energy promotes really fast heating; (3) since microwave energy is
absorbed directly by the solution and not by the vessel walls and vapor
phase, there is a temperature gradient during the first heating stages.
It means that the solution is at a higher temperature than the vessel
walls and gas phase. Consequently, the condensation of formed gases
occurs during the first heating stages and the increase of pressure
is not so abrupt; (4) condensation and the presence of oxygen help
recover nitric acid and it was fully demonstrated that it is possible to
carry out the digestion of organic samples with dilute solutions of nitric
acid, such as 1 or 2 mol/L. The thermal decomposition of hydrogen
peroxide is a good source of oxygen and this reagent also increases
the oxidant power of nitric acid. So, dilute nitric acid solution plus
concentrated hydrogen peroxide is an interesting mixture to be tested
for some samples with high contents of organic compounds; (5) based
on these characteristics of the microwave-assisted heating method,
the consumption of reagents can decrease and can have a substantial
impact on the costs and on the quality of the analytical blank.
31
5. Which are the microwave technologies

available? Which are the pluses and minuses
of each?
MILESTONE Milestone designs and produces all of the

microwave instruments and accessories that
H E L P I N G
you may need for an efficient sample preparation
C H E M I S T S
of pharmaceutical samples. ETHOS UP. The

ETHOS UP (Figure 3) fully embodies Milestone’s philosophy in
microwave sample preparation. Specifically designed for closed
vessel acid digestion, it offers a perfect integration between
microwave hardware, user interface, reaction sensors, and
Figure 3. Milestone ETHOS UP

pressure vessels. The ETHOS UP encompasses Milestone’s
visionary concept of “Total Microwave Sample Preparation”
and, with a comprehensive choice of accessories, it offers
a complete first-class solution also for microwave solvent
extraction, organic and inorganic synthesis, protein hydrolysis
and vacuum evaporation. The new Milestone ETHOS UP
microwave cavity has a volume in excess of 70 L, by far the
largest currently available. Why is this important and what are
the main implications of this design? Firstly, digestion rotors with
more sample places can be accommodated thus improving
productivity and sample preparation throughput. Secondly,
the microwave unit is inherently much safer because a larger
cavity contains more efficiently the gases escaping from vessels,
should there be a sudden over-pressurization. The ETHOS UP
is equipped with two 950 W magnetrons for a total of 1900 W.
The system employs a rotating diffuser that evenly distributes
microwaves throughout the cavity. High power coupled with
the diffuser enables very fast heating of high throughput rotors.
The Milestone ETHOS UP is equipped with the most advanced
yet easy to use reaction sensors (Figure 4) for a complete
quality control of the digestion conditions. Direct temperature
and pressure control are used in a single reference vessel.
Contact-less temperature is available for all vessels. The actual
temperature of each and every vessel is continuously shown
on the instrument control terminal during the microwave run,
allowing an instant visual check of the digestion conditions. In
addition, a contact-less pressure sensor monitors and controls all
of the vessels simultaneously, preventing any leakage or venting.
33
IR SENSOR
DIRECT SENSOR
Figure 4. Direct and contact-less temperature control in all of the vessels
The ETHOS UP features a full stainless steel door with a unique

opening and self-resealing mechanism (Figure 5). Should there
be a sudden over-pressurization of the cavity, the door slightly
opens for a rapid and safe pressure release and the microwave’s
power is instantaneously cut off. Immediately afterward, the
Figure 5. ETHOS UP pressure-responsive door

door is pulled back, resealing the cavity. For additional safety, an
automatic door locking system does not allow the user to open
the ETHOS UP door during the microwave run. At the end of
the run, the door remains locked until the solutions have cooled
down to a user preset temperature. This prevents the misuse
of the instrument and the chemist’s exposure to high pressure
vessels. The ETHOS UP is controlled via a compact terminal
(Figure 6) with an easy-to-read, bright, full-color, touchscreen
Figure 6. ETHOS UP control terminal
display. The terminal is provided with multiple USB and

Ethernet ports to interface the instrument to external devices
and to the local laboratory network. The terminal runs a user-
friendly, icon-driven, multi-language software to provide an easy
control of the microwave run. Simply recall a previously stored
method or create a new one, press ‘START’ and the system
35
will automatically follow the user defined temperature utilizing a

sophisticated PID algorithm. Hundreds of applications, including
all US EPA methods, are preloaded in the ETHOS UP terminal.
There is no need to input the number of samples or weights
being digested, as the software will automatically regulate
the microwave power accordingly. This assures a consistent
quality of the digestion and simplifies the instrument’s use. Two
Figure 7. SK-15 Rotor
Figure 8. MAXI-44 Rotor
completely new rotors, offering both a high quality digestion
and a high sample throughput, are available. The SK-15 (Figure
7) is a high-pressure rotor featuring up to 15 TFM vessels
with a volume of 100 mL and suitable for all applications. The
MAXI-44 (Figure 8) is a high-throughput rotor featuring up to
44 TFM vessels with a volume of 100 mL and suitable for a
wide range of samples including environmental and organic
samples. Both rotors are fully compliant with commonly used
standard methods, such as the US EPA 3015, 3051, and 3052.
The SK-15 and the MAXI-44 feature an enhanced ‘vent-and-
reseal’ technology that controls the inner pressure of all vessels
in complete safety. This patented (US Patent 5,270,010)
technology provides the operator with unsurpassed safety and
performance capabilities: highest temperature and pressure,
highest safety standards, ease of use, and very fast cooling.
Only the excess pressure is released from the vessel. This
ensures both no stress to the microwave system’s door and no
sample loss as it could happen in the case of a membrane or
disk bursting. Finally, a large selection of high purity quartz and
TFM inserts is available for the SK-15 and the MAXI-44 rotors
for smaller sample amounts or to minimize the dilution factor of
the analytical solution.
ultraWAVE. Milestone is committed to the constant pursuit

of game changers in microwave instrumentation. The new
ultraWAVE (Figure 9) exceeds norms and breaks conventions.
The concept of the ultraWAVE was developed by Milestone
years ago, and this technology is now accessible to all analytical
37
Figure 9. Milestone ultraWAVE
laboratories. Milestone’s unique Single Reaction Chamber

(SRC) technology overcomes the limitations of all conventional
microwave sample preparation systems. At the center of the
ultraWAVE is a Teflon-lined 1 L stainless steel reaction chamber,
which serves both as a microwave cavity and as a reaction
vessel. Samples are placed into vials and suitable reagents
Figure 10. Operating sequence

are added. Vials are placed in a rack, which is automatically
lowered into the reaction chamber. The chamber is sealed and
pre-pressurized with inert gas, which physically acts as a cap
for the vials in order to avoid the solutions to boil and to prevent
cross contamination. At the completion of the microwave
run, a built-in cooling device rapidly lowers the temperature
(Figure 10). Available rack configurations include 4, 5, 15, and
22 positions (Figure 11). Vials are available in Teflon, quartz or
disposable glass, and are fitted with loose-fitting Teflon caps to
ensure pressure equalization. Unlike conventional microwave-
assisted digestion systems, no vessel assembly or disassembly
is required and, with disposable glass vials, no cleaning step is
needed. This greatly enhances the ease of use and increases
Figure 11. ultraWAVE rack
39
your sample turn around time. The ultraWAVE vials do not

require any capping tool. A reusable Teflon cap is placed on top
of the vial in a fraction of a second. The ultraWAVE is an ultra
high performance system, operating at a pressure of up to 199
bar and a temperature of up to 300 °C. This allows the complete
digestion of extremely difficult samples and of large amounts of
organics. Unlike all conventional microwave-assisted digestion
systems, every sample is under a direct temperature and
pressure control; there is no need to rely on a reference vessel
or to an indirect control such as infrared temperature sensors.
This assures complete control of the digestion process in every
sample. The ultraWAVE reaches high temperatures faster,
cools down faster, and is capable of working with a higher
pressure and temperature than any closed vessel system. The
ultraWAVE is operated via a compact control terminal with an
easy-to-read, bright, full-color, touchscreen display. With the
Milestone ultraWAVE, any combination of sample types can
be digested simultaneously; there is no need to batch samples
into identical types. No development method is needed, as
the same method can be used for almost every sample type,
and there is no need to use different rotors for different sample
types. And, for the first time, blanks and reference standards
of any matrix can be digested alongside samples, enabling a
true in-run digestion quality control. The ultraWAVE vessel has
a volume of 1 L. This is far bigger than any other microwave
digestion system, where the typical vessel volume is of 25-
100 mL. As a result, along with an allowable pressure of 200
bar, the ultraWAVE is capable of digesting a total amount of
organic samples by far greater than any other device. A total of
20 g of dry weight organics can be digested in a single run; for
example, 4 g per sample, when using the 5-place rack, can be
digested. Compared to all conventional microwave digestion
systems, the ultraWAVE is easier to use and the work flow is
dramatically improved. 15 samples are processed in 45 min
start to finish. Furthermore, the ultraWAVE sample throughput
is far better than sequential microwave digestion systems,
where each sample requires at least 15 min in order to be
prepared. There is no cross contamination among samples in
the ultraWAVE. Furthermore, blanks are significantly lower with
respect to conventional microwaves, since less acid is used and
vials have less surface in contact with the analytical solution.
The determination of the residual carbon content offers a good
understanding of the digestion’s completeness.
6. Is there any general procedure that I could

adopt?
As potential procedures for sample preparation, I would

suggest applying the following steps: (1) think about
the main sample constituents and excipients; (2) if the
sample is not a powder, think about how you could
pulverize it; (3) evaluate if direct dilution with water is applicable; (4)
41
evaluate if direct dilution with organic solvents may be used. According

to Lewen, cited in the references list (please, see Reference list 2),
the solvents often used are dimethyl sulfoxide, 2-butoxyethanol and
dimethylformamide, but methanol, ethanol, and isopropanol may also
be used; (4) evaluate if nitric acid digestion is effective. You may test
concentrated nitric acid (i.e. 14 mol/L) and solutions containing 7 and
2 mol/L; (5) evaluate if hydrogen peroxide may improve nitric acid
digestion; (6) evaluate if aqua regia digestion is effective; (7) evaluate
if inverted aqua regia digestion is effective; (8) certainly there are other
options and it is highly recommended to read Lewen’s paper recently
published in Spectroscopy.
7. Which are the steps that I should follow in

order to establish an analytical procedure for
sample digestion?
It is always important to have information on the samples’

characteristics and on how they were produced. This
will give you indications about possible constituents
and contaminants. Additionally, information on the
sample matrix will give you a good idea on how to design a suitable
sample preparation procedure. Solid samples must be ground and
the use of a ball mill would be appropriate. Normally, you are going
to digest around 200-500 mg of sample and it is important to have
a homogeneous sample for a proper representativity when taking an
aliquot for analysis. Considering the samples’ characteristics, you
may follow the steps suggested in Chapter 2, Q6, and test possible
digestion strategies. A solution containing a maximum of 10% V/V
nitric or hydrochloric acids will be suitable for measurements either
by ICP OES or ICP-MS.
8. How could I improve analytical blanks?
Analytical blanks are a major issue for measuring low

concentrations of target elements. It is very unusual
to contaminate a sample with less common elements,
such as Ir, Os, Pd, Rh, and Ru. On the other hand, it is
relatively easy to contaminate samples with common elements, such
as Cr, Cu, and Ni. Ideally, an analytical blank is a material containing
sample matrix constituents but without any trace of analytes. This
material should be treated as a sample and all of the steps of the
analytical procedure should be applied in order to obtain a blank
solution. This will give the analyst information on the blank levels of
analytes and it is important in order to avoid false positives in the
analysis of real samples. It is well known in analytical chemistry that
an analytical result cannot be better than the quality of the sampling
and of the sample preparation steps. We may say that another critical
requirement, in order to obtain accurate and precise results, is to
have proper control of the analytical blank. Taking into account that
most samples will require the application of microwave-assisted acid
digestion, we must pay attention to the quality of the acids we use. The
use of acids produced by sub-boiling distillation is highly recommended
43
and Milestone has the apparatus for performing acid purification with
minimum involvement of the analyst. Finally, digestion vessels may be
cleaned by adding pure water or pure reagents to them and by running
a heating program without any samples. This is a simple and effective
procedure for cleaning the vessels and to avoid memory effects.
MILESTONE Milestone’s Clean Chemistry Line is an innovative

and complete portfolio of systems and
H E L P I N G
accessories to reduce and control the analytical
C H E M I S T S
blank in ultra-trace elemental analysis. There is a

growing awareness that sample preparation should evolve to
the same standards of the most modern analytical techniques,
such as ICP-MS, and there are a number of factors that can
critically impact the quality of the data:
-- The purity of the reagents
-- The cleanliness of the material in contact with the sample
-- The sample preparation method
Each of these factors is related to the reduction and to the
control of the analytical blank. To address this issue, Milestone
has developed a comprehensive line of products (Figure 12)
and accessories aimed to reduce and control the analytical
blank, which perfectly complement the ETHOS UP and the
UltraWAVE microwave digestion systems.
duoPUR. The chemical reagents used during the analysis

are an important source to the analytical blank. Sub-boiling
distillation has been demonstrated to be the best method of
acid purification. It uses contact-less infrared lamps to vaporize
Figure 12. Milestone’s Clean Chemistry Line
the surface liquid at a temperature typically of 20 °C below the

boiling point. In contrast to conventional distillation, in which
a strong boiling action generating aerosolized particles results
in the contamination of the original liquid with the distillate,
a gentle surface evaporation during sub-boiling distillation
prevents the formation of spray or droplets and yields to a very
high pure acid. The duoPUR consists of two quartz distillation
units. Each unit contains two infrared heating elements, a water
cooled condenser, a high-purity PFA collection bottle, and a fully
automatic acid loading and discharging system. The vaporized
liquid is collected in the inclined water-cooled condenser and
drips into the collection bottle. The microprocessor-controlled
distillation process allows the user to set the distillation time and
power level by using a compact control terminal with an easy-
to-read, bright, full-color, touchscreen display. The distillation
rate ranges from 50 to 400 mL per hour, depending on the
power setting, the cooling water temperature, and the acid’s
boiling point.
45
subCLEAN. The Milestone subCLEAN is a compact and easy-

to-use sub-boiling system, in which all of the parts in contact
with the acids are made of high-purity fluoropolymers. The
subCLEAN is therefore suitable for the purification of HF, as well
as for HNO3 and HCl. The acid is automatically loaded into the
distillation container, where it is gently heated below its boiling
temperature. The entire microprocessor-controlled process
uses a compact control terminal with an easy-to-read, bright,
full-color, touchscreen display. The subCLEAN does not require
cooling water or a chiller, as acid vapors rapidly condense into a
collection bottle by forced air cooling.
traceCLEAN. Cleaning the various items used in ultra-trace

analyses is a critically important laboratory routine. To minimize
contamination, traditional cleaning methods require the items to
be soaked in hot acids, often for several hours. In order to be
effective, large volumes of acid are consumed and need to be
changed regularly. There is also a substantial risk of exposure to
hot acids and acid vapors using traditional soaking techniques. To
address these issues, Milestone has developed the traceCLEAN,
a fully automated, self-contained, acid steam cleaning system
for the accessories used in trace metal analyses. Place the items
that need to be cleaned in the traceCLEAN system, program
the required time and temperature, and then press “Start”.
Freshly distilled acid vapors will continuously reflux within the
sealed unit, thoroughly leaching any metal contaminants from
the items. Various holders are available for vials, microwave
digestion vessels, flasks, glassware, and ICP-MS accessories.
9. How could I evaluate the efficiency of
a digestion process?
For samples mainly containing organic constituents,

the efficiency of the digestion may be established by
determining the concentration of dissolved organic
carbon in the final digest. This measurement can be
performed simultaneously to the determination of other analytes
either by using ICP OES or by using ICP-MS (see the Reference list).
This parameter may be presented as a concentration of dissolved
organic carbon in the final digest and gives us an idea about the
possible interferences in plasma processes. Alternatively, if we know
the original carbon concentration of the sample, we may easily
calculate the residual carbon content (RCC) as a percentage of the
original carbon content and the RCC informs us about the efficiency
of the digestion, i.e. the fraction of organic compounds oxidized.
For samples mainly containing inorganic constituents, it is possible,
depending on the sample matrix, digestion mixture and maximum
digestion temperature, to have solid residues in the final digest. The
presence of solids indicating an incomplete digestion may or may not
be critical. Ask yourself about the sample’s composition, the analytes,
and the digestion procedure and critically evaluate if a fraction of the
analytes may or may not be trapped in these solid residues. A partial
digestion does not necessarily indicate an unsuccessful digestion and,
if possible, the solid residue would be characterized by a technique,
such as the X-ray fluorescence.
47
10. Which are the main specifications and

parameters to consider when selecting the
proper digestion system? Sample, type,
amount, vessel volume?
For samples mainly containing organic constituents,

the choice of digestion mixture is straightforward when
using microwave-assisted heating, and I certainly
recommend using nitric acid plus hydrogen peroxide.
For instance, for 500 mg of sample, you may add 5 mL,
2 or 7 mol/L of HNO3 plus 3 mL of H2O2 concentrated. For samples
mainly containing inorganic constituents, the choice of digestion
mixture is not so simple and you may follow a strategy like the one
discussed in Chapter 2, Q6. In both cases, temperature is the key
factor for a successful digestion and maximum temperatures around
180-220 oC should be enough for most samples. For microwave-
assisted acid digestions, typical heating times will be around 30-40
min. It is worth mentioning that, at a lower temperature, the digestion
will not be effective; if the temperature is high enough, time should
not be a critical issue, unless a too short amount of time is set.
11. What is the role of T and P in a digestion
process?
Temperature is the parameter that we must control in

order to carry out effective digestions. Pressure is only
a side effect resulting from the release of gases in a
closed vessel. The positive aspect is that it increases
the boiling point of the acid mixture; on the other hand, the negative
aspect is that it requires special vessels and devices in order to avoid
over pressurization.
49
CHAPTER 3
DETERMINATION OF ELEMENTAL
IMPURITIES USING ICP OES
51
Determination of elemental impurities
using ICP OES
1. What do you mean by ICP OES?
ICP OES stands for inductively coupled plasma optical

emission spectrometry. I am sure you remember the
first experiments in undergraduate laboratories when
you performed flame tests to qualitatively detect the
presence of metals, such as sodium, lithium, and potassium. You
know you were measuring the radiation emitted by excited atoms
decaying to ground state in a combustion flame kept in a Bunsen
burner. ICP OES is based on the same principle: the emission of
radiation by excited atoms and ions, but using high temperature argon
plasma to provide enough energy to excite and ionize most elements
of the periodic table. Considering the capability of exciting almost all
chemical elements, ICP OES is a typical multielement technique, i.e. it
has the full capability of determining simultaneously, or sequentially, all
elements required by USP <232>, <233>, and <2232>.
53
Chapter 3 - Determination of elemental impurities using ICP OES
2. What is argon plasma?
Based on Bunsen-Kirchhoff’s law, we know that the

population of atoms in excited state increases when
we increase the temperature of the atomizer. This is the
essence of using argon plasma: it provides an elevated
temperature source to excite or even to ionize most elements of
the periodic table, except for halogens and noble gases. The ideal
gas to form a plasma should present the following characteristics:
low chemical reactivity, low background emission, suitable thermal
conductivity, ionization energy, electrical resistance, availability, and
acceptable cost and safe handling. Compared to other possibilities,
argon is the gas that has all of the attributes to form inductively coupled
plasma. The ionization of argon starts by using a Tesla coil and an
alternating electromagnetic field, generated by applying radiofrequency
alternating current (27 or 40 MHz) through an induction coil, which
induces ions to move. Kinetic energy and friction are converted into
heat and a temperature of up to 10,000 K is reached depending on
the plasma region.
3. How is argon plasma generated?
As just mentioned in Chapter 3, Q2, the ionization

of argon starts by using a Tesla coil. Ions move in
response to an alternated magnetic field generated by
an alternated current applied through a copper coil.
The argon plasma is kept at the tip of a torch formed by three quartz
tubes concentrically positioned. Argon gas flows through these tubes,
named outer tube, intermediate tube, and injector tube. Argon flow
rates are controlled independently through these tubes; this flow rate
is typically of around 15 L/min between the outer and the intermediate
tubes. This is called plasma gas and it is important in order to preserve
the plasma and to cool down the outer tube walls, in order for them
not to melt. The auxiliary gas is introduced between the intermediate
tube and the injector tube in flow rates of around 1 L/min and it helps
to spatially fix the argon plasma and to avoid the formation of deposits
in the tip of the injector tube when working with solutions containing
high concentrations of dissolved solids. Finally, the nebulizer, or carrier
gas, flows through the injector tube and transports the sample aerosol
into and through the plasma. Typical nebulizer gas flow rates range
from 0.6 to 1.0 L/min and it must be kept absolutely constant because
it is related to the residence time of aerosol particles in the plasma
and also to the amount of sample introduced into the plasma. It may
be surprising to new users that the total argon flow rate is around 20
L/min with a total of 1200 L in 1 h of operation. However, please do not
forget about the multielement character of ICP OES and its analytical
55
capability to determine around 40 elements in 1 min, depending on

the optical system and on operational conditions.
4. What do I measure in ICP OES?
ICP OES measurements are based on the detection of

emission lines resulting from decay processes that go
from excited levels to the ground state. Ionic lines are
emitted by excited ions and atomic lines are emitted
by excited atoms. Ionic lines are represented by II when related to
monovalent excited ions. Atomic lines are represented by I. Each
excited atom or ion has its own characteristic spectra and we may
choose different lines to perform measurements. The emission
intensity is correlated with the analyte concentration and this is the
important analytical property for quantitative measurements. On the
other hand, the emitted wavelengths are typical of each element and
they provide a qualitative fingerprinting of the respective analyte.
5. How do I choose emission lines?
Argon plasma provides up to 15.7 eV of energy. When

choosing an emission line, we must consider the amount
of energy needed to form the excited species and the
spectral environment around the emitted lines, taking
into account the sample matrix. The sample matrix is composed by
all of the constituents present in the sample, in combination with the
analytes we wish to determine. Spectral interferences are common
processes in ICP OES and we must critically choose lines not affected
by these processes. The control software of each instrument informs
us of the available emission lines for each analyte, their respective
relative intensities and potential spectral interferences. The decision
should take into consideration the sample matrix and, consequently,
the spectral environment. Additionally, each emission line has a
characteristic relative intensity and we can choose either a line
with a high relative intensity when determining an analyte at trace
concentrations, i.e. mg/L or µg/L range, or a line with a low relative
intensity when determining an analyte at higher concentrations.
57
6. Which are the components of an ICP OES?
In a general view, an ICP OES is composed by five

components: (1) a radiofrequency power unit; (2) a
sample introduction system and a quartz torch; (3) a
pre-optical and an optical system; (4) a detector; and
(5) signal acquisition and treatment. The radiofrequency power unit
delivers energy to preserve the argon plasma. The power typically
applied varies from 0.9 to 1.5 kW and the radiofrequency alternating
current oscillates at either 27 or 40 MHz. The sample introduction
system is generally composed by a nebulizer and a nebulization
chamber. The goal here is to convert the liquid sample into an aerosol
with a monomodal distribution of particle sizes. Particles with diameters
of up to 5 µm are carried by the argon nebulization gas (Chapter 3,
Q3) and transferred to the argon plasma. The pre-optical system is the
region of the equipment in which the emitted radiation travels through
the entrance slit towards the optical system. The optical system is a
critical part of ICP OES and it must have a high resolution, i.e. around
8 pm in the ultraviolet region, to separate really close emission lines.
There are different optical arrangements, but most of them are effective
in providing both a high resolution and an elevated transference of
the resolved radiation to the detector. Detectors may also vary, but
solid state detectors are frequently used to provide the possibility of
a fast and simultaneous detection of multiple emission lines for each
element that is being determined. The spectral coverage range varies
typically from 160 to 800 nm and most determinations are carried
out in ultraviolet region. Emission line measurements below 200 nm
require the removal of air due to the absorption caused by oxygen. The
signals acquired are background corrected and a dedicated software
is applied to facilitate the analysis.
7. Which kinds of samples can I introduce in

ICP OES?
Generally, we analyze liquid samples in ICP OES.

Solid samples are usually converted into liquids by
applying microwave-assisted digestion (Chapter
2). Liquid samples must have proper physical and
chemical characteristics in order to be introduced without causing
transport interferences and without deteriorating the components of
the sample introduction system and of the quartz torch. Therefore,
we must consider the total dissolved solids, the acid concentration,
the solution viscosity and density, the type of acid, and the type
of solvent. All these parameters depend on the selected nebulizer,
the nebulization chamber, and the torch position, i.e. vertical or
horizontal. In general, total dissolved solids should be lower than
1% m/V in order to avoid the nebulizer or the central tube to block,
memory effects, and the deposition of salts in the quartz tubes or in
the pre-optical interface. The acid concentration should be lower than
10% V/V and the best acid medium for sample preparation is nitric
acid because when we introduce this acid into the argon plasma,
we are introducing elements (i.e. H, N, and O) already present in the
59
plasma due to air diffusion or to water dissociation. When working

with more viscous acids, such as sulfuric or phosphoric acid, it is
better to keep the maximum concentration around 5% V/V. We may
also introduce samples dissolved in organic solvents, but we must
be careful of the volatility of the solvent, the organic load of argon
plasma and the applied radiofrequency power, which is normally high
in organic media compared to aqueous media. Finally, solutions with
a pH higher than 8 must not be introduced into the ICP quartz torch;
solutions with a high pH contain elevated concentrations of sodium
and potassium that may deposit in the quartz tube and decrease the
lifetime of the quartz torch.
8. How do I operate an ICP OES? Which are the

critical parameters?
The operation of an ICP OES is fully controlled by a

software that allows the analyst to select the operational
parameters, the elements and the emission lines to
be measured, the correction of background signals,
data acquisition, calibration control and the generation of reports.
Frequently, it is also possible to choose the viewing position for each
analyte, which can be axial or radial (Chapter 3, Q9). Each company
developed its own control software and, nowadays, we can say that
they are user-friendly. The most critical operational parameters are the
observation zone, the applied radiofrequency power, and the nebulizer
gas flow rate. The observation zone should be chosen considering
the maximum analyte emission signal and the minimum background
emission. Argon plasma has different temperature zones and
companies recommend specific elements and respective emission
lines in order to set a compromise observation zone. Nowadays, this
is a straightforward parameter that must be adjusted and the analyst
must be aware of its implications. The applied radiofrequency power
is related to the plasma’s temperature and it is typically set around
1.0 to 1.3 kW depending on the sample medium and the complexity
of its matrix. The nebulizer gas flow rate is related to the speed of
the sample’s aerosol transport through the plasma. The sample
amount effectively transferred to the argon plasma is also related to
the particle sizes of the formed aerosol and the sample nebulization
flow rate. These parameters are controlled by the sample introduction
system and by the internal diameter of the propulsion tube used
for the sample aspiration and the rotation speed of the peristaltic
pump. All these parameters must be controlled in order to introduce
a constant amount of aerosol sample into the argon plasma, which
generates intense and stable analytical signals. The typical residence
time of aerosol particles flowing through the argon plasma is around
2 ms and aerosol particles must have diameters smaller than 5 µm
to be able to desolvate, dissociate, atomize, excite, and ionize when
traveling through the plasma in so short time.
61
9. How do you choose between measurements

in axial or in radial position?
Radial position means that the radiation generated in

the transversal axis of the plasma is being transferred
to the entrance slit of the optical system. On the
other hand, axial position means that the radiation
generated in the longitudinal axis of the plasma is being transferred
to the entrance slit of the optical system. At the beginning, all of the
measurements of this technique were performed in radial position with
the quartz torch vertically positioned. Later on, the axial position with
the quartz torch horizontally positioned also became common, but
in order to make this analytically feasible, it was essential to develop
interfaces to remove the cold tail of the argon plasma. This cold tail
must be removed to avoid self-absorption processes and to decrease
the amount of background emission transferred to the optical system.
Developed interfaces were based on either a perpendicular flow of gas
to cut the cold plasma tail, called shear gas interface, or a frontal gas
flowing towards the cold plasma tail, called end-on gas interface. Both
interfaces are effective, but the shear gas interface requires higher flow
rates of an inert gas, such as nitrogen. Since the flow rate in the end-
on gas interface is lower, typically around 1 L/min, argon gas is used
without a considerable increase of argon consumption. Measurements
in axial position are attractive because sensitivities are higher than
those typically obtained when using the radial position. However, the
analyst must be aware of the fact that the interferences may be more
severe when working with complex matrices, i.e. matrices containing
high amounts of dissolved solids. It is also important to keep in mind
that a quartz torch kept in a horizontal position may have its lifetime
reduced due to the formation of salt deposits in the outer quartz
tube. This effect can be attenuated either by diluting the sample or
by increasing the flow rate of the plasma gas. A rule of thumb is that
the axial position is recommended for less complex matrices when a
higher sensitivity is desired and the radial position is recommended
when a higher sensitivity is not mandatory, but interferences may be
critical. In other words, there is a compromise condition between
sensitivity (i.e. how far down you can go in concentration levels) and
robustness (i.e. how ICP OES measurements will deal with potential
interferences in a complex medium).
10. How do I know if an ICP OES is in good

operational conditions?
I think a practical approach to evaluate operational

conditions is to look for figures of merit that allow us to
infer the equipment’s performance. Typical deviations
of ICP OES measurements are around 1-2%. Specific
measurements may indicate the performance of specific components.
For instance, the performance of the sample introduction system may
be evaluated using the Mg I 285.213 nm emission line. Experimentally,
we aspirate a Mg solution, such as 1 mg/L Mg2+ in 1% V/V HNO3,
and we check the relative standard deviation (rsd) for successive
measurements. If the rsd is higher than 2%, we may infer that either
63
the nebulizer, the nebulization chamber, or sample transport through

the plasma, are not working properly. Similarly, we have other simple
experiments in order to evaluate specific compartments. We will not
discuss these experiments in detail, but we do recommend reading
Reference list 12 and 13 in order to understand this topic better.
11. How do I calibrate an ICP OES?
The point to remember here is that ICP OES is a

multielement technique. It means that all elements
requested by new USP regulations can be simultaneously
measured. Calibration is performed using solutions
that contain the combinations of all of the elements that we need to
determine; we usually prepare 5 solutions with a gradual increase in
concentrations. A critical aspect is that the medium used to prepare
these solutions should be the same medium that you have in your
digested and diluted samples; thus, it is important to take into account
the physical and chemical properties of solutions and their effects on
the nebulization process. In other words, you must consider the acids
that you have used for microwave-assisted sample preparation and
the residual amount that has remained in the digested samples. No
worries, a simple acid-base titration gives you this information and
you do not have to repeat this titration for a determined digestion
procedure. Sulfuric and phosphoric acids should not be used in order
to avoid transport interferences. If possible, prepare the samples using
nitric acid, hydrochloric acid, and hydrogen peroxide; these are the
best reagents to introduce using pneumatic nebulization and these
acids can be easily purified using a sub-boiling apparatus (Chapter 2,
Q8). The best alternative, in order to prepare the analytical solutions
for calibrating ICP OES, is to buy multielement stock solutions
provided by several producers and to dilute these according to the
desired calibration range. Remember that ICP OES has a large linear
range and analytical solutions can cover a large concentration range
without losing the linear correlation between each analyte’s emission
intensities and element concentration.
12. Which kinds of interferences may I expect?
Interferences can be classified as transport, spectral,

and chemical processes. Transport interferences
are related to the sample introduction system and
the critical point here is to calibrate solutions that are
chemically and physically similar to the samples. It means that the
analyst must be careful with the procedures adopted for sample
preparation and the reference solutions to be calibrated must simulate
the chemical medium of the digest solutions. Spectral interferences
are common in ICP OES because the high temperature of the argon
plasma excites almost all of the chemical elements and each element
may emit several wavelengths. The control software informs the
analyst about the typical interferences for each emission line and,
65
based on the sample’s information, the analyst must make judicious

choices. In other words, based on the sample’s main constituents, the
analytes to be determined, the available emission lines, and the inter-
element effects, the analyst must think about the spectral environment
and he or she can choose non-interfered emission lines in order to
perform accurate and precise determinations (Chapter 3, Q5). Due to
the elevated temperature of argon plasma, interferences caused by
chemical processes, such as the formation of refractory compounds,
are not so common; however, pay attention to classic processes, such
as the presence of high concentrations of easily ionized elements (e.g.
Li, Na, and K) and high concentrations of elements such as carbon,
sulfur, and phosphorus.
13. How do I solve these interferences?
Transport interferences are solved by matrix matching

the chemical and physical characteristics of reference
solutions and samples. Transport interferences are also
corrected by adding an internal standard (Chapter 3,
Q14) to reference solutions and samples. The choice of a simple and
effective microwave-assisted digestion procedure is an important step
in order to avoid transport interferences. As already mentioned, the
use of sulfuric and phosphoric acids should be avoided because of
the viscosities of these reagents. Spectral interferences are common
in ICP OES considering the huge number of emission lines for each
analyte. Nowadays, several instruments combine an optical system
and a solid state detector that allow the analyst to choose from
several different wavelengths for each analyte. The analyst can choose
the best line taking into account sensitivities and also by carefully
evaluating the spectral environment around a specific emission line.
Chemical interferences are not so common in ICP OES due to the
high temperature of the argon plasma. However, as just mentioned
in Chapter 3, Q12, pay attention to the effects caused by easily
ionized elements, such as Na and K, which perturb the ionization
equilibrium when working with ionic lines. These processes can be
controlled by matrix matching chemical media for reference and
sample solutions. Dissolved organic carbon may also cause effects
on specific elements, such as As and Se. In this case, the adoption of
an efficient microwave-assisted sample preparation method is highly
recommended. Some samples have a complex medium considering
all concomitants and, in this case, it may be tricky to matrix match
the chemical media of these samples with reference solutions. In this
situation, operating the argon plasma in robust conditions may help
circumvent interferences. It means that aerosol particles will travel
through the plasma for a longer period time and the plasma will be
kept at a higher temperature. Robust conditions are reached by
decreasing the nebulization gas flow rate (i.e. lower than 1.0 L/min) and
by increasing both the diameter of the central tube of the quartz torch
(i.e. around 2 mm) and the radiofrequency applied power (i.e. around
1.4 kW). However, robust conditions should be adopted only when
working with complex samples because self-absorption processes
may increase and the linear calibration range may decrease.
67
14. What is an internal standard?
As you may already know, this strategy is commonly

adopted in chromatographic measurements. An
internal standard is an element, not originally present
in the sample, that should be added to all reference,
blank, and sample solutions and its signal should be measured
simultaneously to the analyte’s signals. All of these solutions should
have the same element concentration selected as the internal
standard. The choice of an internal standard is not straightforward and
the analyst must carefully think about the type of interference that he
or she wants to correct, the behavior of the analytes, and the internal
standard in argon plasma. When using an internal standard, the
analyte concentration is related to the ratio between the analyte signal
and the internal standard signal. This strategy allows the correction of
certain interferences and it is particularly effective to correct transport
interferences during sample introduction by pneumatic nebulization.
15. What should I expect when applying ICP
OES to USP <232>, <233> and <2232>?
A modern ICP OES should have a suitable performance

in the determination of most elements required by USP
<232>, <233>, and < 2232> and some studies are
demonstrating this capability (see the Reference list).
However, depending on the sample digestion conditions and ICP OES
conditions, a lack of sensitivity may occur to some trace elements, such
as As, Hg, and Pb. Literature has also demonstrated memory effects for
Os. These elements should be carefully checked when developing an
analytical procedure for specific pharmaceutical samples. In general,
to attend USP <232>, <233>, and <2232> regulations, we expect
the application of ICP OES to be relatively simple and the analytical
throughput will be determined by the selected procedure for sample
preparation (Chapter 2).
69
CHAPTER 4
DETERMINATION OF ELEMENTAL
IMPURITIES USING ICP-MS
71
Determination of elemental impurities
using ICP-MS
1. What do you mean by ICP-MS?
ICP-MS stands for inductively coupled plasma mass

spectrometry. In this technique, instead of measuring
the radiation emitted by excited atoms and ions, we
focus our attention on the formation of ions in the
argon plasma. A specially designed interface transfers these ions
to a mass spectrometer in order to measure the analytes based on
their mass/charge ratios. Taking into account both the temperature
of argon plasma and ionization energies, it is known that, for most
chemical elements, the major species present in argon plasma are
the monovalent cations. Similarly to ICP OES, ICP-MS is also a
multielement technique and it has the full analytical capability to
determine all of the elements required by USP <232>, <233>, and
<2232>. The sensitivity of ICP-MS is typically of 103 fold higher than
that usually obtained using ICP OES and determinations in µg/L
range, or even ng/L, are commonly performed if sample preparation
was carried out using clean procedures and the analytical blank was
fully controlled.
73
Chapter 4 - Determination of elemental impurities using ICP-MS
2. Is this argon plasma the same one used

in ICP OES?
Yes, it is the same argon plasma and the discussion

presented in Chapter 3, Q2 and Q3, remains valid.
Good news! That means that you already know what
you need.
3. What do I measure in ICP-MS?
As just mentioned in Chapter 4, Q1, in ICP-MS, the

ions generated in the argon plasma are transferred by
an interface towards the mass spectrometer and then
to the detector. For most chemical elements, here
generically represented as M, the main species in the argon plasma is
M+ and the mass/charge ratio for unitary charge is equal to the mass
of the natural isotopes of each element.
4. How do I choose isotopes?
Each chemical element has one or more natural isotopes

and we consider the abundance of these isotopes in
order to select the ones we will measure. For instance,
Cr has four natural isotopes: 50Cr with an abundance of
4.345%; 52Cr with an abundance of 83.789%; 53Cr with an abundance
of 9.501%; and Cr with an abundance of 2.365%. To be able to
54
reach the best sensitivity, we should make measurements using the

Cr+ isotope; however, the determination of this isotope is affected by
52
the spectral interference caused by 40

Ar12C+ and we must eliminate
or correct this process. Strictly speaking, these species do not have
the same mass/charge ratio, but a quadrupole mass spectrometer
(Chapter 4, Q5) does not have enough resolution to separate them.
Fortunately, there are alternatives. For instance, we may improve
sample preparation and decrease the RCC (Chapter 2, Q9) as much
as possible. We may also use special instrumental arrangements that
allow us to destroy the molecular ion 40Ar12C+. Finally, if we do not want
to cope with this process, we may choose a different isotope, e.g.
Cr+. Some chemical elements are monoisotopic, such as 27Al, 59Co,
53
As, etc. In this case, we must establish the instrumental conditions

75
that allow us to solve typical interference processes, such as the well-

known effects of 40Ar35Cl+ on 75As+ in samples containing chloride ions.
Just to illustrate other similar situations to Cr, let us consider the 6
natural isotopes of Se: 74Se with an abundance of 0.89%; 76Se with an
abundance of 9.36%; 77Se with an abundance of 7.63%; 78Se with an
75
abundance of 23.78%; 80Se with an abundance of 49.61% and 82Se

with an abundance of 8.73%. In this case, Se is frequently determined
by using isotope 78 in order to avoid the interference of 40Ar40Ar+ on
80
Se+. The same chloride ions, mentioned above for As, also have
an effect on the determination of V. Just as food for thought, think
about the 2 natural isotopes of V: 50V with an abundance of 0.250%
and 51V with an abundance of 99.750%. How does chloride affect the
measurement of 51V+? (Tip: remember that argon plasma also contains
other atomic species, such as H, O, and N).
5. Which are the components of an ICP-MS?
Typically an ICP-MS is composed by: (1) a radiofrequency

power unit; (2) a sample introduction system and a quartz
torch; (3) an interface region; (4) a mass spectrometer;
(5) a detector; and (6) signal acquisition and treatment.
In general terms, the discussion previously presented for ICP OES
remains valid here (Chapter 3, Q6). However, the interface region and
the mass spectrometer are distinctive characteristics of ICP-MS and
they will be briefly discussed here. Ions are generated in an argon
plasma operated at ambient pressure and must be transferred to
the mass spectrometer and to the vacuum operated detector. The
interface region is composed by a sampler cone, a skimmer cone,
and electrostatic lenses for a gradual pressure decrease between the
plasma and the mass spectrometer and, consequently, to allow the
transference of ions. The sampler cone is positioned in the argon plasma
tip and together with the skimmer cone and the electrostatic lenses
they represent the pathway of ions towards the mass spectrometer.
To allow a gradual pressure reduction and to avoid blockage, cone
orifices usually have internal diameters that range from 0.4 to 1.2 mm.
Because of these small orifices, the solutions introduced into the ICP-
MS should contain a maximum of 0.1% m/V of dissolved solids; when
preparing the sample, it is important to take into account the sample
mass, the amount of reagents added to the digestion, and the final
dilution of the digest. This is not a critical limitation considering the
superb sensitivities provided by ICP-MS. The separation of ions, based
on their mass/charge ratios, is generally performed using a quadrupole
mass analyzer with a practical resolution of 1 atomic mass unit. An
ICP-MS with a mass spectrometer, designed with double-focusing
magnetic sector technology, is available when a high resolution is
required. However, an ICP-MS equipped with a quadrupole mass
analyzer is fully compatible with USP requirements.
6. Which kinds of samples can I introduce in

ICP-MS?
Similar to ICP OES, we generally analyze liquid samples

in ICP-MS. Again, solid samples are usually converted
into liquids by applying a microwave-assisted digestion
(Chapter 2). All of the observations made on ICP OES
remain valid for ICP-MS (Chapter 3, Q7). The quartz torch in ICP-MS
77
is always horizontally positioned. This torch position, and mainly the

small orifices of sampler and skimmer cones, generally implicates that
the completely dissolved solids should be lower than 0.1% m/V in order
to avoid the nebulizer or the central tube to block, memory effects,
and the deposition of salts in the quartz tubes or in the interface of the
cones. Consider this aspect when delineating a sample preparation
procedure.
7. Which are the critical parameters when

operating an ICP-MS?
There are several operational parameters that must

be adjusted when using ICP-MS. Fortunately, several
parameters, such as the formation of double charge
species, the formation of oxides, and applied voltages
in the electrostatic lenses can be adjusted by using a well designed
control software and each company provides recommendations
on how to set optimum operational conditions. The three most
important parameters that the analyst must control are: (1) the applied
radiofrequency power; (2) the nebulization gas flow rate; (3) and the
sampling depth, i.e. the distance between the argon plasma and
the orifice of the sampler cone. The applied radiofrequency power is
related to the plasma temperature. The nebulization gas flow rate is
related to the residence time of aerosol particles in the plasma and,
consequently, affects the formed species. These two parameters
affect the type of species that will be predominant in the plasma
and, together with the sampling depth, have a strong influence on
the species that are being transferred to the mass spectrometer in
order to reach the detector.
8. How do I know if an ICP-MS is in good

operational conditions?
When initiating an ICP-MS procedure, we must check

sensitivities for low, medium, and high mass ranges.
There is a default sensitivity that is expected for each
isotope and this value checks isotopes in the three
mass ranges. Usually, the formation of double charged species is
checked by measuring the Ba2+/Ba+ ratio and the formation of oxides
is checked by measuring the CeO+/Ce+ ratio; both parameters should
be as low as possible. Average values are below 2%. The equipment’s
stability may be checked by measuring one reference solution during
different operation times and by comparing the obtained signals. It
is also highly recommended to use certified reference materials to
evaluate the accuracies of all the determined elements.
79
9. How do I calibrate an ICP-MS?
As in multielement techniques, the calibration is

performed using reference solutions containing several
analytes. The acid medium must be compatible with
the one you have made for sample preparation; in
other words, pay attention to the acid type and concentration in
the final digests. Due to the space charge effect (Chapter 4, Q10),
ICP-MS signals are strongly affected by matrix components and it
is essential to work with matrix-matched reference solutions and
samples. If the sample matrix is too complex and cannot be kept
in the reference solutions without critical contamination issues, the
calibration could be performed by applying the standard additions
method. Please revise it.
10. Which kinds of interferences may I expect?

How do I solve them?
Transport interferences are the same ones already

discussed for ICP OES (Chapter 3, Q12). Notice that
ICP-MS measurements are affected by the space charge
effect. Sorry to be specific, but I must explain this. As
we have seen in Chapter 4, Q5, ions are transferred from the plasma
to the mass spectrometer through the interface. Due to the gradual
decrease of both temperature and pressure in the interface region,
ions tend to spread when being transported through the interface.
Electrons are lighter than any other ion and move faster. A charge
imbalance will cause repulsion between cations in the ion cloud. Heavy
ions exert a strong repulsion in order to remove lighter ions. So far, we
do not know how to control these effects in the interface region and,
to be able to compare the signals generated by the analytes present
in the samples and in the reference solutions, we need to matrix
match these solutions to be able to equalize space charge effects.
As just mentioned in Q9, the use of the standard additions method
may be needed. Spectral interferences occur when species with close
mass/charge ratios are not separated by low-resolution quadrupole
mass spectrometers. Typical examples are 35
Cl16O+, Ar35Cl+, and
40
Ar12C+, respectively affecting the determination of

40 51
V+, As+, and
75
Cr+. The simplest alternative to be able to solve these interferences

52
is to select a different natural isotope to make the measurements.

Sometimes, this is neither feasible, e.g. As that only has one natural
isotope, nor attractive, e.g. V that has two natural isotopes with
completely different relative abundances, i.e. 50V with 0.250% and 51V
with 99.750%; consequently, measuring V at a mass/charge of 50,
implies an appreciable loss of sensitivity. Alternatively, we may improve
sample preparation and chloride may be removed in order to avoid
interferences on As and V; or, carbon may be completely oxidized
using a microwave-assisted digestion to destroy carbon compounds
and to avoid effects on the determination of Cr at its main isotope at
a mass/charge of 52. Special instrumental arrangements may also be
used.
81
11. What is an internal standard?
As mentioned before in Chapter 3, Q14, internal

standards are effective to correct transport
interferences. Additionally, space charge effects
(Chapter 4, Q10) for ICP-MS measurements may also
be corrected by adding an internal standard, or standards, to the
calibration solutions and samples. An internal standard should be
chosen by considering its mass, its behavior in argon plasma, and
its purity. All of these aspects are critical for ICP-MS applications
and must be carefully considered. A rule of thumb is that the internal
standard should have an atomic mass as close as possible to the
atomic mass of the analyte.
12. What should I expect when applying ICP-MS

for USP <232> and USP <233>?
An ICP-MS should carry out a suitable performance in

the determination of all the elements required by USP
<232>, <233>, and < 2232> and some studies are
demonstrating this capability (see the Reference list).
The analyst must pay special attention to the spectral interferences;
the selection of the natural isotopes to be measured for each analyte
should take into account this aspect and the relative abundances.
Operational conditions should be carefully set and special devices,
such as collision cells, reaction cells, and tandem mass spectrometers,
may be needed in order to solve specific effects on the analytes,
particularly those with natural isotopes with a mass/charge ranging
from 40 to 80 u.m.a.. As previously mentioned for ICP OES (Chapter
3, Q15), we may expect the successful application of ICP-MS to
attend USP <232>, <233>, and <2232> regulations and analytical
throughput will be determined by the selected sample preparation
procedure (Chapter 2).
83
CHAPTER 5
VALIDATION PROCEDURE
85
Validation Procedure
1. How could I validate developed analytical

procedures?
USP <233> presents the requirements for the validation

of an elemental impurities procedure for both limit tests
and quantitative determination procedures. It is highly
recommended to read this material together with the
Validation of Compendial Procedures <1225>. In this chapter, we
will briefly comment the figures of merit that should be checked for
the validation of procedures. We also suggest reading USP 730 on
plasma spectrochemistry.
2. How could I evaluate accuracy?
It is recommended to test accuracy by performing spike

recovery studies. Thus, standard solutions containing
the target elements, at concentrations ranging from 50
to 150% of J, should be prepared for testing. The USP
87
Chapter 5 - Validation Procedure
defines the J value for each element according to its permissible daily
exposure (PDE), maximum daily dose (MDD), and dilution factor (DF),
considering the sample preparation procedure: J = PDE / MDD x DF.
Samples must be spiked before performing any sample preparation
step at concentrations ranging from 50 to 150% of J for each target
element. The acceptance criteria establishes spike recoveries that
range from 70 to 150% for three replicates at each concentration.
3. How could I evaluate precision?
USP <233> recommends to test repeatability by

using six independent samples of test material taken
from the same lot spiked with the target elements at
the indicated level. The relative standard deviation for
each target element, for the six individual sample determinations,
should be lower than 20%. It is also pointed out that the evaluation of
ruggedness should be performed by repeating the analysis on different
days, or with different instrumentation, or with different analysts, or
combinations thereof. The relative standard deviation for each target
element should be lower than 25%.
4. How could I evaluate sensitivity?
The sensitivity of each target element is directly

calculated by the slope of the analytical calibration curve
for ICP OES and ICP-MS, i.e. by the variation of the
element’s analytical signal in function of the analytical
concentration.
5. How could I evaluate specificity?
According to USP <233>, “the procedure must be

able to unequivocally assess each target element in
the presence of components that may be expected to
be present, including other target elements, and matrix
components”. Pay attention to matrix effects on ICP OES and ICP-
MS measurements and set operational conditions in order to overcome
these effects. The occurrence of matrix effects can be evaluated by
making measurements using solutions prepared with different masses
of known matrix constituents. Alternatively, measurements for sample
solution digests under different dilutions, and also additions and
recoveries of target analytes may allow inferences on the matrix effects.
89
Chapter 5 - Validation Procedure
6. How could I evaluate sample throughput?
Sample throughput is related to the number of samples

that can be analyzed per hour by applying the developed
analytical procedure. Multielemental techniques, such
as ICP OES and ICP-MS, have a superb analytical
capability of conducting fast and sensitive measurements. Therefore,
sample throughput will be determined by the procedure developed
for sample preparation and microwave-assisted procedures will be
an important strategy in order to reach a performance compatible
with the measurement requirements of emission lines and of isotopes
using argon plasmas.
CHAPTER 6
FINAL REMARKS
91
“So, my dear friend, how do you feel
now about your new USP task? Do you
still see it as a big challenge?”.
“Not really. I do thank you for all your

advice. I feel more confident. Of
course, there are a lot of points that I
need to grasp, but now I know the
fundamentals and I am more prepared
to start the development of the
analytical procedures to attend new
USP regulations. Fortunately, I feel like
I have the support to be able to answer
any further questions”.
93
Chapter 6 - Final Remarks
“Great. However, please think about

trace analysis when implementing new
USP regulations. Sometimes it is easy to
contaminate samples and you must pay
attention to the sample preparation step,
since it is known to be the critical step
when considering contaminations and
losses. In fact, USP recommends the
use of closed vessels when carrying out
digestions. Do not forget that the quality
of the results is critically dependent on
your sample preparation step. As we
have already discussed, a microwave-
assisted sample preparation using closed
vessels gives you trustable analytical
procedures. Finally, I suggest you to
consider our discussion as a starting
point and it is highly recommended to
read the material cited in the references
list”.
APPENDIX A
95
References
1. R. C. Richter, J. A. Nóbrega, C. Pirola, Think Blank. Clean Chemistry

Tools for Atomic Spectroscopy. 1st ed., Sorisole, Milestone, 2016.
2. N. Lewen, Preparation of pharmaceutical samples for elemental impurities
analysis: some potential approaches. Spectroscopy, 31(4):36-43,2016.
3. R. Thomas, Replacing traditional heavy metals testing with modern plasma-
based spectrochemical techniques. Spectroscopy, 31(3):24-33,2016.
4. S. T. Gouveia, F. V. Silva, L. M. Costa, A. R. A. Nogueira, J. A. Nóbrega,
Determination of residual carbon by inductively coupled plasma optical
emission spectrometry with axial and radial viewing, Analytica Chimica
Acta, 445:269-275,2001.
5. C. D. B. Amaral, R. S. Amais, L. L. Fialho, D. Schiavo, A. R. A. Nogueira, J.
A. Nóbrega, Determination of carbon in digested samples and amino acids
by inductively coupled plasma tandem mass spectrometry, Microchemical
Journal,122:29-32,2015.
6. J. A. Nóbrega, C. Pirola, L. L. Fialho, G. Rota, C. E. K. M. A. de Campos
Jordão, F. Pollo, Microwave-assisted digestion of organic samples: how
simple can it become?, Talanta, 98:272-276,2012.
7. C. D. B. Amaral, L. L. Fialho, F. P. R. Camargo, C. Pirola, J. A. Nóbrega,
Investigation of analyte losses using microwave-assisted sample digestion
and closed vessels with venting, Talanta, 160:354-359,2016.
8. J. Nölte, ICP Emission Spectrometry. A Practical Guide. 1st ed.,
Weinheim, Wiley-VCH Verlag, 2003.
9. M. Rury, The importance of method development for trace element
analysis by inductively coupled plasma-optical emission spectrometry,
Spectroscopy, 31(5):16-32,2016.
10. J. L. Todoli, J. M. Mermet, Liquid Sample Introduction in ICP
Spectrometry. A Practical Guide. 1st ed., Amsterdam, Elsevier, 2008.
11. R. Thomas, Practical Guide to ICP-MS. A Tutorial for Beginners. 3rd
97
Appendix A
ed., Boca Raton, CRC Press, 2013.

12. J. M. Mermet, E. Poussel, ICP Emission Spectrometers: 1995 analytical
figures of merit, Appl. Spectrosc., 49(10):12A-18A,1995.
13. F. V. Silva, L. C. Trevizan, C. S. Silva, A. R. A. Nogueira, J. A. Nóbrega,
Evaluation of inductively coupled plasma optical emission spectrometers
with axially- and radially-viewed configurations, Spectrochimica Acta,
Part B, 57:1905-1913, 2002.
14. H. Wiltsche, M. Winkler, P. Tirk, Matrix effects of carbon and bromine in
inductively coupled plasma mass spectrometry, J. Anal. At. Spectrom.,
30:2223-2234,2015.
15. Agilent, Elemental Impurity Analysis in Regulated Pharmaceutical
Laboratories, 2015.
16. Thermo Scientific, The Medicine Maker. Determining Elemental
Impurities in Pharmaceutical Products and Dietary Supplements: A
QA/QC Primer for USP <232>, <233>, <2232> and ICH Q3D, 2015.
17. J. S. Barin, P. A. Mello, M. F. Mesko, F. A. Duarte, E. M. M. Flores,
Determination of elemental impurities in pharmaceutical products and
related matrices by ICP-based methods: a review. Analytical and
Bioanalytical Chemistry, 408:4547-4566,2016.
AUTHORS
99
Authors
Joaquim A. Nóbrega received his Ph.D.

from the State University of Campinas in
1992 and completed his postdoctoral training
with Prof. Ramon Murray Barnes (University
of Massachusetts, Amherst, MA, 1996) and
with Prof. Bradley Todd Jones (Wake Forest
University, Winston-Salem, NC, 2003). He
is a Full Professor at the Department of
Chemistry, Federal University of São Carlos
(São Carlos, São Paulo State, Brazil) and is a
Visiting Professor at the Faculty of Pharmacy,
University of Concepción (Concepción,
Chile). His main research interests are sample
preparation in inorganic analysis, atomic
absorption spectrometry, atomic emission
spectrometry, and inductively coupled plasma
mass spectrometry. He is a member of the
Group of Applied Instrumental Analysis.
In 2011, he co-authored a chapter on
“Microwave-Assisted Sample Preparation
for Spectrochemistry” published in the online
Encyclopedia of Analytical Chemistry (John
Wiley & Sons) and in 2014, he co-authored
a chapter on “Diluted Acids in Microwave-
Assisted Wet Digestion” published in the book
“Microwave-Assisted Sample Preparation for
Trace Element Determination” (Elsevier).
Camillo Pirola is the Marketing and
Business Development Manager at Milestone.
After receiving his degree in Chemistry,
in May 1999, Camillo joined Milestone as
an Application Chemist. His professional
career interlaces with Milestone’s overall
growth. He then became Application
Manager, providing application support to all
Milestone distributors around the world. In
the late 90’s, he became Area Manager for
several countries including Japan, Italy, The
United Kingdom, Spain, Russia, India, and
Brazil. While retaining his position of Area
Manager, in the mid-2000’s, Camillo became
responsible for the Milestone marketing at
a global level. Since 2012, he is also the
Business Development Manager at Milestone.
Camillo has contributed to several scientific
papers and lectures, and is often invited to
give presentations, seminars, and training
courses worldwide.
101
GETTING READY FOR
USP 232, 233 AND 2232
Are you ready for new United States Pharmacopeia 232, 233, and 2232
and also ICH Q3D pharma regulations considering inorganic elemental
impurities? We hope yes, but maybe you are not. Frequently pharma
laboratories are a world of chromatographs used for organic analysis.
Now we are moving for a room with inductively coupled plasmas with
optical emission or mass spectrometers (you will get used to new
acronyms: ICP OES and ICP-MS). Yes, you will have new analytical
instruments in your lab. Together with ICPs, you will also be engaged
in sample preparation. You will use new procedures for as complete as
possible oxidation of organic compounds and destruction of excipients
followed by the determination of elemental constituents liberated in the
liquid phase. Microwave-assisted preparation in closed vessels will
become an important ally. This is the topic of this book where two
analysts talk about this new demand and how to get prepared for it. A
Q&A style was adopted for making your reading easier and funnier. The
authors hope you enjoy it.

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J.A. Nóbrega - C.

GETTING READY FOR

GETTING READY FOR

Edited by: Milestone Srl and Ikonos Srl

“Work may be hard sometimes. I

“Trust me. It is not as hard as you think”.

“Let’s make a plan. Organize your

OVERVIEW OF USP <232>, <233>

1. I am used to working with USP <231> and

Figure 1. Typical car in the early 1900s

As you know, USP <231> - Heavy Metals was issued

limit specified in individual monograph in percentage (by weight)

2. How would you summarize USP <232>?

USP <232> Elemental Impurities – Limits specifies the

3. How would you summarize USP <233>?

USP <233> - Elemental Impurities - Procedures

4. Could you please also inform me on what the

Good point. ICH stands for International Conference

The elements included in the Q3D Guideline were

inherent toxicity. This information was directly obtained from the

6. How would you summarize USP <2232>?

USP <2232> deals with the determination of elemental

7. How can I prepare my laboratory in order to

Laboratories of pharmaceutical industries are proficient

8. What are the main difficulties that I should

ICPs are certainly ready for the determination of

Chapter 4, Q10. Sample preparation is usually the most critical step

9. Which are the key concepts that must be

I would say that the inorganic elemental analysis,

It is difficult to discuss a standard timetable that will vary

1. USP <232> recommends the use of a

Sample preparation in closed vessels presents several

2. Which are the reagents that I should consider

Sample digestion should be carried out using reagents

Working with concentrated acids is potentially critical

Figure 2. Organic sample mass vs developed pressure

4. Do you recommend the use of microwave-

USP <233> recommends the use of a closed vessel

5. Which are the microwave technologies

MILESTONE Milestone designs and produces all of the

of pharmaceutical samples. ETHOS UP. The

Figure 3. Milestone ETHOS UP

32 GETTING READY FOR USP 232, 233, AND USP 2232

Figure 4. Direct and contact-less temperature control in all of the vessels

The ETHOS UP features a full stainless steel door with a unique

Figure 5. ETHOS UP pressure-responsive door

34 GETTING READY FOR USP 232, 233, AND USP 2232

Figure 6. ETHOS UP control terminal

display. The terminal is provided with multiple USB and

will automatically follow the user defined temperature utilizing a

Figure 7. SK-15 Rotor

Figure 8. MAXI-44 Rotor

ultraWAVE. Milestone is committed to the constant pursuit

Figure 9. Milestone ultraWAVE

laboratories. Milestone’s unique Single Reaction Chamber

Figure 10. Operating sequence

38 GETTING READY FOR USP 232, 233, AND USP 2232

Figure 11. ultraWAVE rack

your sample turn around time. The ultraWAVE vials do not

6. Is there any general procedure that I could

As potential procedures for sample preparation, I would

evaluate if direct dilution with organic solvents may be used. According

7. Which are the steps that I should follow in