1. business and industrial
2. pharmaceutical industry

APPLICATION REPORT
Sample Preparation
Revised USP chapter for
elemental impurities
Revision of USP <231>
Application of closed vessel
digestion for pharmaceutical
samples
The United States Pharmacopeia (USP) has
developed a new test for elemental impurities
in drug products which will be official in mid of
2013. The new general chapter <232> (Limits)
and <233> (Procedures) will replace the current USP <231> (metallic impurities). The revised chapters present new procedures, e.g.
closed vessel digestion, for the evaluation of
analytes. The following paper intents to explain
how Berghof digestion systems comply with the
requirements of the new method.
Introduction
Controlling and monitoring of metal impurities in pharmaceutical samples (e.g. final drug, raw material, API, excipients,
catalyst) has outstanding importance. Metal contaminants do
normally not have any therapeutic effect, but might lead to
severe side-effects and affect the stability of drugs.
Currently, inorganic impurities are tested by an almost 100year
old qualitative method which is described in USP General
Chapter <231>. The method bases on a colorimetric test which
detects only metals precipitating by sulfide ions (Pb, Hg, Bi, As,
Sb, Sn, Cd, Ag, Cu, Mo). To comply with the heavy metals limit,
the colored precipitate has to be compared visually with a 100
ppm Pb standard. Further disadvantages of USP <231> are:






No quantification of individual concentration of selected
elements
Several elements of interest are not considered
(e.g. platinum group elements (PGE), Cr, Ni)
Inaccurate results due to visual comparison
Loss of volatile elements (e.g. Hg) due to ashing step
(600°C) for solid samples
Costly and time consuming
Non-specific and insensitive
APPLICATION REPORT | SAMPLE PREPARATION
S. 2/6
Industry criticized that USP <231> is not in accordance with
current requirements. Therefore, USP started to revise USP
<231> to USP <232>/<233> (drug products), and USP <2232>
(dietary supplements). The new methods are intended to be
more accurate, reliable, specific and robust.
Even if the new general chapter will be official of February 1st,
2013 the implementation is expected to be finished middle of
the year.
dissolved in an appropriate solvent to enable analysis. If the
sample is not dissolvable, digestion techniques should be applied. Suitable digestion techniques should be chosen according
to sample composition, analytes of interest and their concentration. Possible methods are as well closed-vessel digestion
techniques and open-vessel procedures. But, it is stated that in
case of digestion of volatile elements, open-vessel methods are
not recommendable.3 To identify the right method a system
suitability test should be performed which includes:
Harmonization with Ph. Eur.
The International Conference on Harmonization of Technical
Requirements and Registration of Pharmaceuticals for Human
Use (ICH) is aiming to develop a global policy to uniformly
regulate limits of metal impurities in drug products and ingredients for the three big economic areas Europe, USA and Japan.
For this, bit by bit general methods are adapted with regards to
harmonization.
Analogues to USP <231>, Ph. Eur. describes methods for heavy
metals testing by using thiocetamide reagent in chapter 2.4.8.
In order to harmonize the methods according to ICH guidelines, Ph. Eur. published new chapters 5.20 (Metal catalyst or
metal reagent residues) and 2.4.20 (Determination of metal
catalyst or metal reagent residues). However, methods described in USP and Ph. Eur. are comparable but not yet fully
harmonized. While USP requests analysis of 15 elements
(Cd, Pb, As, Hg, Ir, Os, Pd, Pt, Rh, Ru, Cr, Mo, Ni, V, Cu)1,
Eur.Ph. only requests 14 elements (Ir, Os, Pd, Pt, Rh, Ru, Cr,
Mo, Ni, V, Cu, Mn, Fe, Zn)2. Furthermore, Ph. Eur. gives a
5 years’ timeline for the implementation of the method which
will be finished 1st September 2013.1


In General Chaper 5.20 EMA Guideline “on specification limits
for residues of metal catalysts or metal reagents”
(EMEA/CHMP/SWP/4446/2000) is implemented. With regards to the ongoing harmonization process ICH Q3D guideline
(Impurities: Guideline for metal impurities) is going to replace
the EMA guideline introduced in this chapter. General text 5.20
is aimed to recommend exposure and concentration limits for
metal catalyst and metal residues present in pharmaceutical
substances (API, excipient) or drug product. However, limits
are based on classification of metal residues in three categories
depending on their risk to human health.2



Class 1: Metals of significant safety concern
(Pt, Pd, Ir, Rh, Ru, Os, Mo, Ni, Cr, V)
Class 2: Metals with low safety concern (Cu, Mn)
Class 3: Metals with minimal safety concern (Fe, Zn)
Spike recovery experiment
Clear solution as acceptance criteria for sample digestion
Limits specified in USP <232>
USP requires that all elemental impurities (catalyst and environmental contaminants) occurring in drug substances, drug
product or excipients have to comply with the specified limits.
Especially As, Cd, Pb, and Hg have to be controlled due to their
ubiquitous occurrence and toxic potential for humans.
Thereby, As limits are based on the most toxic form – the inorganic As. Method of choice are techniques determining the total
As content under the assumption that the As present in the
sample is inorganic. Nevertheless, if the value exceeds the limit
individual methods for speciation have to be applied to differentiate between inorganic and organic form.
Limits for Hg are based on the inorganic Hg2+ which is more
abundant in pharmaceuticals than the more toxic methyl mercury. However, limits for substances with potential of containing methyl mercury are defined in the corresponding monograph.
Permissible daily exposure (PDE) limits defined in general
chapter <232> are valid for a larger number of elements (incl.
PGE). Furthermore, elemental impurity limits base on the
toxicity which is directly related with the bioavailability of elements.
The table below depicts the allowed values for elemental impurities for drug products. It is important to note that limits have
to be adjusted according to the type of drug and the route of
administration. For example, Cd limits for parental or inhalational administration must meet a PDE which is 10 times lower
than the oral administration. Moreover, large volume parental
(LVP) components have set limits 100 times lower than the oral
administration.
For determination of the elements the use of a validated quantitative method is recommended (semi-quantitative test of
metal sulfides as described in Ph. Eur. 2.4.8 is not suitable).
Ph. Eur. Chapter 2.4.20 is not providing a specific method for
sample preparation since composition and specification limits
of substances are differing. Solid samples are requested to be
L_USP233_53-0215-97-00-01-001, Subject to changes and errors, Printed in Germany
APPLICATION REPORT | SAMPLE PREPARATION
S. 3/6
Elemental Impurities for Drug Products1
Elements
Oral
Parental
Inhala-
LVP
Daily
Daily
tional Daily
Compo-
Dose
Dose
Dose
nent Limit
[µg/day)
[µg/day]
[µg/day]
[µg/day]
Cd
25
2.5
1.5
Pb
5
5
5
0.5
Inorg. As
1.5
1.5
1.5
0.15
Inorg. Hg
15
1.5
1.5
0.15
Ir
100
10
1.5
1.0
Os
100
10
1.5
1.0
Pd
100
10
1.5
1.0
Pt
100
10
1.5
1.0
Rh
100
10
1.5
1.0
Ru
100
10
1.5
1.0
Cr
*
*
25
*
Mo
100
10
250
1.0
Ni
500
50
1.5
5.0
V
100
10
30
1.0
1000
100
70
25
Cu
0.25
Procedures specified in USP <233>
General chapter <233> includes two analytical procedures
(ICP-AES, ICP-MS) for determination of elemental impurities
in pharmaceuticals.
However, due to the broad range of drug substances and products a single approach suitable for all sample types is not applicable. Sample pretreatment techniques should be individually
chosen according to the substance. It is distinguished between
neat, direct aqueous solution, direct organic solution and indirect solution.4 If a pretreatment technique is not indicated in
the monograph, analysts are asked to follow the depicted decision tree for choosing a suitable procedure.
* not a safety concern
Now, for drug substances and excipients individual concentration limits are available. The acceptable level of elemental impurities depends on the ultimate use and has to be determined
by the manufacturer. The following table presents values of
drug products dosed at a maximum daily dose of <10 g/day.
The values serve as default concentration limits allowing quality control in the supply-chain for drug substances and excipients.
Concentration Limits for Drug Substances and Excipients1
Element
Concentration
Concentration
Concentration
limit [µg/g] for
limit [µg/g] for
limit [µg/g] for
oral drug
parental drug
inhalational
products
products
drug products
Cd
Pb
2.5
0.25
0.15
0.5
0.5
0.5
Inorg. As
0.15
0.15
0.15
Inorg. Hg
1.5
0.15
0.15
Ir
10
1.0
0.15
Os
10
1.0
0.15
Pd
10
1.0
0.15
Pt
10
1.0
0.15
Rh
10
1.0
0.15
Ru
100
10
1.5
Cr
*
*
2.5
Mo
10
1.0
25
Ni
50
5.0
0.15
V
100
10
30
Cu
100
10
7
* not a safety concern
Elemental impurities decision tree 5




Neat: suitable for liquids, or unsolvated samples
Direct aqueous solution:
sample soluble in aqueous solvent
Direct organic solution:
sample soluble in organic solvent
Indirect solution: sample is not soluble neither in aqueous nor in organic solvent, closed-vessel procedure using
concentrated acids is recommended
Closed-vessel digestion
A wide range of drug samples will be insoluble in aqueous or
organic solvents. In this case, USP <233> specifies the use of
closed-vessel digestion with concentrated acids but does not
mandate a specific methodology. Since the digestion success
depends on the sample composition and matrix it is up to every
L_USP233_53-0215-97-00-01-001, Subject to changes and errors, Printed in Germany
APPLICATION REPORT | SAMPLE PREPARATION
individual laboratory to develop and validate a suitable closedvessel digestion method (incl. acid mixture and digestion protocols). Nevertheless, USP <233> proposes a method which
turned out to be practical for a variety of samples.
S. 4/6
particularly for laboratories that process only with a limited
number of samples.
“Dehydrate and predigest 0.5 g of primary sample in 5 mL of
freshly prepared concentrated acid. Allow to sit loosely covered
for 30 minutes in a fume hood. Add an additional 10 mL of
concentrated acid and digest, using a closed vessel technique,
until digestion or extraction is complete. Repeat if necessary by
adding an additional 5mL of concentrated acid.4”
The ubiquitous nature of Hg makes it to one of the most important analytes that have to be determined. But, Hg is also well
known to be chemically unstable at low concentration. Therefore, USP <233> indicates to use an appropriate stabilizer
when analyzing Hg.
Digestion of most difficult samples with Berghof`s pressure digestion system
Open vessel methods are widely-used in routine analysis. Nevertheless, to minimize contaminations and to enable the total
digestion of the sample, closed-vessel methods are more advantageous. Closed-vessel methods not only minimize the risk of
losing volatile elements, but also allow higher digestion temperatures. Customers benefit from highly reliable instruments
providing reproducible results in every digestion step.
Berghof digestion systems comply with requirements of
pharmaceutical industry
USP <233> requires closed-vessel digestion methods if the
sample is not soluble in aqueous or organic solvents. Since
many pharmaceutical products are a combination of different
components (API, coating, filler) the solubility is limited. To
destroy completely the matrix, digestion methods with strong
acids are necessary. Here, closed-vessel digestion methods
(especially microwave digestion) are the preferred technique.
Berghof offers closed-vessel digestion systems that are superior
for a broad range of samples in ultra-trace analysis.
Classical closed-vessel pressure digestion systems
Samples with high net-weigh or most difficult samples requesting high temperatures or long digestion times, respectively, can
be digested with Berghof’s pressure digestion system. The system composes of stainless steel pressure digestion vessels with
high quality TFMTM-inliner. The vessels are available in a variety of capacities ranging from 50-250 mL, a maximum operating temperature of 260 °C, and a maximum operating pressure
of 200 bar. In any case, safety is ensured by appropriately dimensioned rupture discs. The isolating nature of the TFMTMinliner leads to slow and gentle heating of the samples. For
safety reasons, heating takes place in a special heating block
enabling the ability to extend the digestion period nearly indefinitely. The pressure digestion system offers a very high
level of flexibility and represents an economical alternative,
Closed-vessel microwave digestion
Due to the complex matrix of pharmaceutical substances and
products, microwave digestion will be the preferred method in
most cases. In Principle, sample solutions are directly heated
by absorbing microwave radiation. This results in extremely
fast and simultaneous heating.
All microwave digestion systems from Berghof are characterized by the extremely durable pressure vessels made of isostatically pressed TFMTM-PTFE. This enables a safe and easy handling. Isostatically pressed TFMTM-PTFE improved material
properties in contrast to conventionally produced PTFE vessel.
For example, surface structure of TFMTM-PTFE is verifiable
smoother with reduced microporosity leading to negligible
contamination effects. Therefore, Berghof pressure vessels
enable reproducible and accurate results even for Hg determination.
The mature vessel concept allows tool-free opening/closing of
the vessels and enables easiest handling.
Moreover, digestion vessels of Berghof are included in the
guarantee and are not regarded as a consumable. Customers
benefit by reduced operative costs. The vessel portfolio ranges
from standard vessels with an operating pressure of 40 bar
with max. 230 °C up to high pressure vessels up to 100 bar and
260 °C.
L_USP233_53-0215-97-00-01-001, Subject to changes and errors, Printed in Germany
APPLICATION REPORT | SAMPLE PREPARATION
S. 5/6
Pressure vessels are made completely of TFMTM-PTFE and consist of only
few parts that can be opened and closed without additional tools.
High flexibility with Speedwave four
Microwave digestion systems are required to be safe and to
deliver reproducible and fast results. Due to the rapid heating,
attended by an increase of the pressure, reaction parameters
have to be monitored constantly during the digestion. For
safety reasons it is recommendable not only to control temperature but also pressure. In Berghof systems both data are
used for precise power control leading to reproducible heating
curves and reproducible digestion results.
The determination of volatile elements according to USP <231>
was almost impossible since samples had to be treated at
600 °C in a muffle furnace. Modern closed vessel digestion
techniques enable the determination of volatile elements without loss of analytes. Berghof digestion vessels are self-sealed by
using lip seal rings. Time-consuming closing with extra-tools
can be refrained. Furthermore, lip sealing lead to hermetically
closed vessels, preventing blowing of volatile elements. The
higher the pressure the tighter the seal. Customer benefit from
safe digestions delivering reproducible results also for volatile
elements.
The unique contact-free temperature measurement speedwave
DIRC® enables real-time temperature measurement in all vessels without using a reference vessel. This effectively increases
the safety of the digestion system. Additionally, Berghof offers
the contact-less optical pressure control speedwave four OPC®
for controlling the internal pressure. The application of contact-less sensor techniques facilitates the handling and reduces
operational costs. Additional sensors in reference vessels are
not necessary anymore.
The unique top-loading concept of speewave four simplifies
crucially the handling. The rotor is fixed in the oven and only
the vessels are individually introduced. Due to isolating behavior of TFMTM-PTFE no active cooling of the microwave is necessary. After the digestion, vessels can be removed from the
oven and externally be cooled (e.g. water bath) which significantly reduces cooling down phase. With this time-saving procedure customer benefit by higher sample throughput.
Berghof digestion systems are ideal for (ultra) trace analysis of
pharmaceutical samples. Pharmaceutical laboratories request
instrumentations complying with strict quality regulations to
ensure security, reproducibility, simplicity and traceability of
the datas.
 Berghof offers installation and operational qualification
(IQ | OQ) documents upon installation
 Restricted user area with password protection
 Record control including storage and traceability of all
individual datas
Recovery determination in pharmaceutical drugs
Berghof digestion systems were tested for their applicability for
drug substances and drug products according to new USP
chapter. Analysis has been performed of As, Hg, Pb and Cd
since these elements are of special importance. For doing so, a
plant drug (Alchemillae vulg) was spiked with Pb, Cd, and Hg
standard solution. A spiked 500 mg sample of Alchemillae vulg
was digested with HNO3:HCl:H2O2 (3.0 mL:0.5 mL:2.0 mL) in
the microwave digestion system speedwave four by using the
following temperature program. The elements have been analyzed by ICP-MS.
L_USP233_53-0215-97-00-01-001, Subject to changes and errors, Printed in Germany
APPLICATION REPORT | SAMPLE PREPARATION
S. 6/6
Temperature program microwave digestion speedwave four
Parameter
Temperature
Step 1
Step 2
Step 3
175
190
210
Hold Time
10
7
30
Rise Time
2
2
5
50
60
80
Power
Recovery of Pb, Cd, Hg in plant drugs
Element
Recovery [%]
Standard deviation [%]
84.5
1.74
Cd
94.9
1.94
Hg
104.7
1.44
Pb
Furthermore, pharmaceutical raw materials and products have
been tested. For this, different pharmaceuticals (e.g. saccharose) were spiked with Pb, Cd, Cr, Ni, and As standard solution.
300-500 mg of sample were digested in a microwave digestion
system with H2O:HNO3:HCl (5:3:0.5) using following temperature program. The analysis was performed by ICP-MS.
Temperature program microwave digestion speedwave four
Parameter
Temperature
Step 1
Step 2
175
190
Hold Time
10
25
Rise Time
10
5
Power
50
80
USP chapter <233> defines acceptance criteria for spike recovery values of 70-150 % for the mean of three replicated preparations. All analyzed elements meet the given requirements with
excellent accuracy. It was shown that Berghof digestion systems
are suitable instruments for sample preparation of pharmaceuticals giving good results in accordance with USP general chapter <233>.
Conclusion
Even if the new methods are more stringent and complex, they
will have a lasting effect on industry. With USP <232> and
<233> a modern test procedure based on present technologies
is available which will lead to higher quality pharmaceutical
products.
Pharmaceutical industry demand instruments being able to
completely digest a broad range of different matrix. The modern instrumentation design enables Berghof digestion systems
to ensure safe and reproducible digestion results complying
with updated requirements stated in USP and Ph. Eur..
The new proposed methods will be more stringent and challenging for laboratories also demanding new analytical instruments. However, customer can benefit from Berghof`s almost
40 years experience in the field of closed-vessel digestion.
Recovery of Pb, Cd, Cr, Ni, As in pharmaceuticals
Element
Recovery [%]
Standard deviation [%]
Pb
95.5
1.73
Cd
95.4
1.25
Cr
98.2
1.27
Ni
97.2
1.41
As
95.6
1.21
1
European Pharmacopeia 7.7.; 5.20. Metal catalyst or metal reagent residues, 04/2013:52000
2
European Pharmacopeia 7.7.; 2.4.20. Determination of metal catalyst or
metal reagent residues, 04/2013:20420
3
Second Supplement to USP 35-NF 30; <232> Elemental Impurities –
Limits, Update January 2013.
4
Second Supplement to USP 35-NF 30; <233> Elemental Impurities –
Procedures, Update January 2013
5
Pharmacopeial Forum, Vol. 36(1), Jan-Feb. 2010
Berghof Products + Instruments GmbH | Harretstrasse 1 | 72800 Eningen | www.berghof.com
L_USP233_53-0215-97-00-01-001, Subject to changes and errors, Printed in Germany