USP 735 as an Alternative to USP 233 for Elemental Impurity Analysis in Pharmaceutical Products

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USP <735> as an Alternative to USP <233> for
Elemental Impurity Analysis in
Pharmaceutical Products
Justin Masone, Dan Davis;
Shimadzu Scientific Instruments, Columbia, MD

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What is the USP?
United States Pharmacopeial Convention
 Non-profit organization that sets the standards for the identity, strength, quality,
and purity of medicines, food ingredients, and dietary supplements.
 USP drug standards are enforced in the US by the FDA

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Current Regulations
USP <231>: HEAVY METALS is the
current chapter regarding elemental
impurities.
It is based on precipitation of the
metal sulfide in a sample and
compares the intensity to a lead
standard.

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New Regulations: Why?
RTI has described <231> as a “non-specific colorimetric test with low
sensitivity requiring significant quantities of the tested material.” 1
Prone to error
Requires skilled analyst to interpret the color correctly
USP <231> omission will become official on January 1, 2018
1 https://www.rti.org/pubs/aaps2013-harrington-poster.pdf

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New Regulations
Chapter <232>:
Chapter <233>:
Chapter <735>:
January 1, 2018
Elemental Impurities - Limits
Elemental Impurities - Procedures
X-Ray Fluorescence Spectrometry
May 1, 2015

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USP <231>
Metals Included:
 Pb, Hg, As, Cd, Sb, Sn, Bi, Ag, Cu, Mo [10]
Three Different Procedures:
 Method I: for substances that yield clear, colorless, preparations
 Method II: for substances that do not yield clear, colorless preparations
under Method I, or interfere with the precipitation of metals buy sulfide ion,
or for fixed and volatile oils (Method II does not recover Hg)
 Method III: wet-digestion method used when neither I nor II can be used

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USP <231> Shopping List
 H2SO4
 Pb(NO3)2
 NH4C2H3O2
 HCl
 HNO3
 NH4OH
 CH3COOH
 Drug Sample
 Grams of sample = 2/(1000*L); where L is the Metals Limit (in %)
 200 mg (Cu)  13.3 g (As)
 C2H5NS
 C3H8O3

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USP <231> Determination
“Filter…into a 50-mL color-
comparison tube…view downward
over a white surface: the color of the
solution from the Test Preparation is
not darker than that of the solution
from the Standard Preparation, and
the color of the solution from the
Monitor Preparation is equal to or
darker than that of the solution from
the Standard Preparation.”
1870
1 http://en.wikipedia.org/wiki/Colorimetry_(chemical_method)
1

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USP <232> Elemental Impurities: Limits
<232> specifies limits for the amounts of elemental impurities (“EI”) in
drug products
EI includes catalysts and environmental contaminants that may be
present in drug substances, excipients, or drug products.
When EI are known to be present, have been added, or have the
potential for introduction, compliance to the levels in <232> is required
If manufacturers can demonstrate the absence of EI, further testing is
not needed

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When testing is done to demonstrate compliance, the “Big Four” must
be included as a minimum (As, Cd, Pb, Hg).
<232> limits do not apply to excipients or drug substances (except
where specified).
<232> limits do not apply to products intended only for veterinary use
and conventional vaccines.
<232> limits do not apply to dietary supplements and their ingredients
(addressed in <2232> Elemental Contaminants in Dietary
Supplements).

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Toxicity limits are defined as maximum permissible daily exposure
(PDE). These differ between routes of exposure, such as:
 Oral (incl. mucosal and topical)
 Parenteral
 Inhalational
 Large-volume parenteral (LVP, 100 mL)
PDE values are in μg/day
 Based on an “average” 50 kg (110 lbs) person
LVP for injection volume >100 mL
 Must be controlled through the individual components used to manufacture
the drug product (Summation Option)

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Element [15] Oral PDE (μg/day)
Parenteral PDE
(μg/day)
Inhalational PDE
(μg/day)
LVP Compondent
Limit (μg/g)
Cadmium 25 2.5 1.5 0.25
Lead 5 5 5 0.5
Inorganic arsenic 1.5 1.5 1.5 0.15
Inorganic mercury 15 1.5 1.5 0.15
Iridium 100 10 1.5 1.0
Osmium 100 10 1.5 1.0
Palladium 100 10 1.5 1.0
Platinum 100 10 1.5 1.0
Rhodium 100 10 1.5 1.0
Ruthenium 100 10 1.5 1.0
Chromium -- -- 25 --
Molybdenum 100 10 10 1.0
Nickel 500 50 1.5 5.0
Vanadium 100 10 30 1.0
Copper 1000 100 100 10

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<233> describes two analytical
procedures for the evaluation of the levels
of EI:
 ICP-OES
 ICP-MS
<233> also allows for an equivalent alternative technique, and describes
certain acceptance criteria.
<233> Elemental Impurities: Procedures

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Sample Preparation
 Forms of sample prep include:
 Neat (unsolvated)
 Direct aqueous solution (sample dissolved in aqueous solvent)
 Direct organic solution (sample dissolved in organic solvent)
• May requires sample introduction system for organics or
additional gases (for example, mixed Ar/O2)
 Indirect solution (digestion)
• Requires microwave digester

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Sample Analysis
 Calibration:
• Matrix-matched blank
• Low standard (0.5J)
• High standard (2J)
 Sample batch
 Blank
 SST (High standard)
 Criteria for pass: ± 20%

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Alternate Procedure Validation
Detectability
Standard solution
• At concentration J
Spike Sample 1
• Analysis sample spiked at J
• Acceptance criteria: ± 15%
Spike Sample 2
• Analysis sample spiked at 0.8J
• Acceptance criteria: I < Spike Sample 1
<233> Elemental Impurities

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Precision (Repeatability)
6 analysis samples
 Independent samples spiked at J
Acceptance criteria
 RSD ≤ 20%

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Accuracy
Spike Samples
 Spike samples at concentrations ranging from 50%-150% of J
Acceptance Criteria
 70%-150% for the mean of three replicate preparations at each
concentration

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USP <735> X-Ray Fluorescence Spectrometry
Chapter <735> describes XRF, and it is not limited to <232>
Elemental Impurities
What is XRF?
Can it satisfy <232> limits?
How does it compare to ICP or ICP-MS?

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First, some clarification…
X-Ray Fluorescence Spectrometry
Energy-Dispersive XRF
(EDXRF)
Wavelength-Dispersive XRF
(WDXRF)
USP <735> X-Ray Fluorescence Spectrometry

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What is ED-XRF?
Energy-Dispersive X-Ray Fluorescence
Energy-dispersive: Ability to discern the energies of x-rays
X-Ray: Form of energy; source of ionizing radiation
Fluorescence: Phenomenon of absorbing energy (short λ) and
subsequently emitting energy (longer λ)

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What are X-Rays?
X-rays are a kind of electromagnetic energy.

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How Do X-Rays Interact with Matter?
When X-rays strike matter, some of them are absorbed and some pass
through.
The degree of absorption and penetration depend on the elemental
composition, density, and thickness of matter.
A consequence of absorption is that secondary X-rays are generated, which
are characteristic of that matter.

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How Do X-Rays Interact with Atoms?
+++++
+
-
-
-
-
- -
-
-
++
-
-
-
-
-
-Irradiating
X-ray
Ejected
Electron
Fluorescent
X-ray

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How Does an EDX Work?
EDX is a type of spectrophotometer. It contains:
X-ray tube
Filters
Collimator
C-MOS
camera
Detector
(SDD)
Sample

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Auto-sampler
12-position auto-sampler
Designed to fit standard
XRF cups
Can run a different
method for each position

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Analytical Range

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USP <735> Shimadzu White Paper
Report to the pharmaceutical elemental analysis method using the
EDX-7000
1. Purpose:
Evaluation of calibration curve and sensitivity of
metal element in solution and cellulose using
EDX-7000
2. Measurement Condition:
The apparatus used for the measurement is
EDX-7000.
The measurement condition is shown in Table 1.

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3. Sample Preparation:
Elements to be measured are Cr, Ni, As, Ru, Pd and Pt.
The sample was prepared by using diluted AAS standard solutions as shown below in
Table 2.

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4. Experimental Condition:
The experimental conditions of each element are shown in Table 3.
5. Result:
5.1 Calibration Curve Evaluation
A 6.0 mL solution and 2.0 g pressed cellulose powder were used for creating
calibration curves of each element. The “RMS” and “Correlation” were
evaluated for reliability of the calibration curves. The calibration curves for the
solution and cellulose of each element are given in below Figures 1 – 12.

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RSD
6.37%
1.39%
2.53%
1.47%
6.18%
3.02%
5.2 Measurement Result at Repeatability Test:
Tables 6 and 7 demonstrate the 10-time repeatability using a solution and cellulose
sample to calculate the average and standard deviation. The sample was used
5.0 ppm for Cr, As, Ru, Pd, Pt, and 30 ppm for Ni.
RSD
5.56%
2.99%
1.21%
4.55%
11.57%
2.61%
Solution
Cellulose

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5.3 Lower Limit of Detection Evaluation:
Tables 8 and 9 demonstrate the lower limit of detection for solution and cellulose
sample from the 10 times repeatability of the blank sample. The lower limit of detection
is defined as three times value of the standard deviation obtained from the repeat
measurement of blank samples.

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5.4 Small Mass Sample Measurement:
100 mg of a cellulose sample was put into the sample cup the calibration curves
and LLDs were evaluated determined and evaluated.
5.4.1 Calibration Curve Evaluation:
100 mg of cellulose powder was used for creating calibration curves of each
element using the conditions in Table 10. The “RMS” and “Correlation” were
evaluated for reliability of calibration curve.

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5.4.2 Low Limit of Detection Evaluation
Table 12 demonstrates the lower limit of detection for a cellulose sample from the
10-time repeatability of the blank. The lower limit of detection is defined as three
times value of the standard deviation obtained from the repeat measurement of
blank samples.

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USP <233> or USP <735>?
Which is the optimal technique?
XRF ICP

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USP <233> or USP <735>?
Application
 What are the detection limits?
 What concentration ranges are expected? (Dynamic range)
 How much accuracy and precision are required?
 How many samples and at what frequency? (Sample throughput)
 How much time spent on sample prep?
 Is there a limited amount of sample available?
 Will matrix interferences have a major impact on analysis?
Installation
 What is the size of the instrument?
 How much lab space is required?
 How clean must the lab/sample prep area be?

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USP <233> or USP <735>?
User
 How easy is the instrument to use?
 What training is required?
Financial
 What is the cost of the instrument?
 What is the cost to run the instrument?
 What is the cost of a specialized laboratory?
 What is the cost to have a dedicated expert(s) to run the instrument?

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USP <233> or USP <735>?
Application
What are the detection limits?
ICP and ICP/MS will have better LODs than EDX. However, this is not of a
concern for USP. ICP/ICP-MS involves sample digestion, which results in
~500-fold dilution of the sample.
EDX requires minimal sample prep, if any at all, and this will not result in any
dilution. EDX meets the limits presented in <232>.
What concentration ranges are expected? (Dynamic range)
Multiple dilutions will need to be performed for ICP/ICP-MS. EDX range is <1
ppm – 100%.

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USP <233> or USP <735>?
How much accuracy and precision are required?
Not a concern; ICP/ICP-MS/EDX all meet USP requirements
How many samples and at what frequency? (Sample throughput)
ICP/ICP-MS have greater capacity for sample throughput (3 minute analysis;
240-position auto-sampler vs. 12-sample turret for EDX).
This may not be that important for many customers, and sample prep may limit the
number of samples per batch for ICP/ICP-MS.
Will matrix interferences have a major impact on analysis?
Interferences with EDX are well-known and easily accounted for (all software based;
post-run). ICP would require a skilled analyst and extra solutions.

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USP <233> or USP <735>?
How much time spent on sample prep?
EDX has the obvious advantage. Sample prep is minimal (pressing sample into a
pellet) to none.
ICP and ICP-MS sample prep could include digestion, multiple dilutions (aqueous or
organic solvents), etc.
This is also an indirect cost of ownership for an ICP-MS—the more samples you have
to prep, the more time is spent paying a highly-trained analyst to do benchwork
chemistry—time that could be spend analyzing other samples (think contract lab).
Is there a limited amount of sample available?
This could be a concern for ICP/ICP-MS (multiple digestions and dilutions to get
samples into working range of instrument). EDX can meet criteria for <232> using only
0.1 g of sample.

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USP <233> or USP <735>?
Installation
What is the size of the instrument?
45 kg (99 lbs)
450 mm x 590 mm x 360 mm
210 kg (462 lbs)
1300 mm x 660 mm x 720 mm
vs.
vs.

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USP <233> or USP <735>?
How clean must the lab / sample prep area be?
EDX is measuring ppm-level, so cleanroom is not necessary.
ICP-MS will require a VERY clean lab and VERY clean prep room.

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USP <233> or USP <735>?
User
How easy is the instrument to use? What training is required?
EDX is the simplest of all elemental analysis techniques.
It can be set up and optimized during installation, only requiring the analyst to
place a sample in the chamber and press start.
ICP-MS is the most complex of all elemental analysis techniques. It requires a
skilled analyst and lengthy training.

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USP <233> or USP <735>?
Financial
What is the cost of the instrument?
EDX: <$75,000. No special accessories required for USP. If anything, a pellet
press could be used ($10,000-$15,000). Total cost is less than $100,000.
ICP-MS: $200,000. Microwave digester ($75,000). Temperature-controlled
spray chamber ($10,000). Total cost is nearing $300,000.
What is the cost of a specialized laboratory?
Not required for EDX. Required for ICP-MS ($50K? $100K?).
ICP/ICP-MS requires ventilation.

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USP <233> or USP <735>?
What is the cost to have a dedicated expert(s) to run the instrument?
Major concern for ICP-MS
What is the cost to run the instrument?
EDX: No consumables. 120V, 2A ($950 over 10 years, including PC)
ICP (~14,450/year)
 Gases (liquid argon)
 Electric ($750/year)
 Consumables (Sample introduction accessories, sample and skimmer
cones, etc.)

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USP <233> or USP <735>?
What is the cost to run the instrument?
EDX cost/sample over 10 years, including:
• Main body
• X-ray tube (2 replacements)
• Inverter
• Detector
• Electric
• For 100 analyses: $0.15/sample
• For 1200 analyses: $1.85/sample
ICP-MS cost/sample over 10 years:
• Industry estimate of RUNNING COST (not including main body and
accessories) is $0.71/sample.
• $2.25/sample including cost of instrumentation

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USP <233> or USP <735>?
Pros Cons
• Minimal to no sample prep
• Cost of the unit
• Cost of ownership
• No gases
• No exhaust
• 120V vs 220V
• Minimal bench space
• No sample waste
• Skilled analyst not required
• Interferences well-
understood and easily
accounted for
• Limited number of samples
(12 vs 240)
• But remember…for
ICP/ICP-MS you will need
to pay someone to prep
the 240 samples first.
• LODs (but not a concern for
USP)
Bottom-line: Cheaper, simpler, more efficient

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Thank You
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http://www.ssi.shimadzu.com/
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USP 735 as an Alternative to USP 233 for Elemental Impurity Analysis in Pharmaceutical Products

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USP 735 as an Alternative to USP 233 for Elemental Impurity Analysis in Pharmaceutical Products

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