Heavy Metals (USP<231>) Revisions
New Limits and Procedures for Elemental Impurities in Pharmaceuticals and Dietary Supplements
By Jeff Grindstaff and Colleen Schroeder
Changes to heavy metals test procedures for the analysis of pharmaceuticals and dietary supplements are under review with new standards set to be in place by mid-2013.4 The intention of the review is to update current analytical testing historically performed using United States Pharmacopeia (USP) <231>. The revisions (USP<232>, USP<233>, and USP<2232>) are designed to set safer limits for public exposure and to reduce the environmental impact of dated methods. Many in the pharmaceutical industry have concerns about the new instrumentation, more stringent requirements, and the associated costs. Nonetheless, the revisions should have a beneficial impact on the industry by significantly improving specificity and analyte recoveries, as well as by yielding overall time savings resulting in safer, higher quality products.
Shift from Outdated Technology to Modern Methodology
First introduced over 100 years ago, USP<231> is a colorimetric procedure based on the precipitation of insoluble metal sulfides. The test is qualitative rather than quantitative. It is not an element specific method, nor is it equally sensitive to each metal. The limits specified by the test are based on the ability to observe the precipitate, rather than on the analysis of toxicological data. The procedure does not necessarily detect all potential forms and/or valences of elements of concern when they are present as the oxo ions or in the organometallic form. Chromium and nickel are potential contaminants from modern stainless steel processing equipment and are not detected by USP<231>. Other studies indicate inconsistent recoveries of monitor and standard solutions using USP<231> method II.2, 3
Industry criticism of this dated method began around 15 years ago and sparked the revision process by the USP. After seeking public comment and advice from experts on metals toxicology, the USP is now recommending that USP<231> be revised to USP<232>, which will require the use of updated instrumental technology to improve selectivity and sensitivity. The change includes modification to the preparation and analysis methodology as well as the impurity limits of each analyte.
Revisions to the elemental impurities test will constitute a serious change for the pharmaceutical industry. The change will shift the testing from a relatively inexpensive procedure that requires minimal set-up and operator training to tests that require expensive instrumentation and highly skilled metals analysts. However, by employing modern instrumental methods, the USP’s intent is to ensure safer products for the consumer as well as offer flexibility and efficiency during testing.
All drug products produced and sold in the U.S. will have to comply with the limits set by USP<232>, and drug substances and excipients will have to be tested and reported for elemental impurities. Likewise, all nutraceutical products will have to comply with limits set by USP<2232>, which includes guidelines for speciating organic and inorganic forms of various elements. USP<232>, USP<233> and USP<2232> are currently in a preliminary recommendation stage and the limits described have not been finalized.
Improved Methodology for Identifying Discrete Elements
One of the main criticisms of USP<231> has been the inability of the testing to recover and identify individual elements. Previously, the elemental impurity list included arsenic, antimony, bismuth, cadmium, copper, lead, mercury, molybdenum, silver and tin due to reactivity of these metals with the sulfide ion utilized in the procedure. The metals were reported inclusively as “heavy metals” due to the procedural inability to show them discretely. In addition, arsenic, bismuth, and molybdenum were not necessarily detected by USP<231> due to common occurrences of these elements in forms inert to the mechanism in the procedure. Since numerous instrumental procedures have been developed over the life of USP<231> that incorporate significant improvements in selectivity and sensitivity, the USP’s proposal will require individual quantification of arsenic, cadmium, lead and mercury (target elements considered most toxic to humans and the environment, see Table 1). If the presence of additional metals is suspected (for instance, if used in the manufacturing process as catalysts or if detected during previous testing), then those additional metals would be added to the target list. Each element screened will have individually distinct impurity limits, based on unique toxicity data.5,6
|Table 1. Health Risks Associated with the Four Elements of Primary Concern5,6,12,13|
|Arsenic (As)||Inorganic forms of arsenic are particularly toxic and water-soluble inorganic arsenic is readily absorbed by the human digestive system. Symptoms include stomach and intestine irritation and skin disturbances, lung irritation and decreased white and red blood cell production. Very high exposure to inorganic arsenic can cause infertility, skin disturbances, declined resistance to infections, heart disruptions, brain damage, and death. Acute oral LD50 values range from 10-300 mg/Kg.|
|Cadmium (Cd)||Cadmium is more readily absorbed through the lungs than through the human digestive system. Exposure to cadmium can damage kidneys, the central nervous system and the immune system, as well as cause bone fractures and reproductive problems. Symptoms can include stomachaches, diarrhea and vomiting. Oral LD50 values in animals range from 63 to 1125 mg/Kg.|
|Lead (Pb)||Exposure to lead can occur through ingestion and inhalation. No clear threshold has been established for lead; however, the USP is deferring to the FDA maximum allowable level for lead in bottled water (5μg/L) to set the elemental impurities limit. Lead can cause: disruption of the biosynthesis of hemoglobin, anemia, high blood pressure, kidney damage, reproductive/fertility problems and brain/nervous system damage.|
|Mercury (Hg)||Prevalence of mercury in the environment leads to biomagnification in the food chain. Organic forms of mercury, such as methyl mercury, are more toxic than inorganic forms due to the ease of absorption into the human system. Symptoms of mercury poisoning include: kidney damage, disruption of the nervous system, damage to brain functions, DNA and chromosomal damage, allergic reactions, sperm damage, birth defects and miscarriages. LD50 values are as low as 1 mg/kg1 in small animals.|
The USP is considering many factors to decide which elements will be tested and at what levels. The likelihood of contamination during manufacturing, possible additional environmental exposure, as well as reactions with other metals (co-exposure) during drug administration are factors influencing the review. Though rapid, accurate, simultaneous multi-element analysis of many metals is now possible at very low concentrations, the USP has preliminarily decided to base impurity limits on toxicologically relevant data in an effort to avoid burdening the industry with unnecessarily low limit requirements. The new limits will be based primarily on previously established guidelines for human and animal toxicological exposure and are dependent on route of delivery (see Table 2). Screening will be required for all toxic metals that have been shown to be present, regardless of whether or not they are included in the impurities list. However, the USP will not mandate methodology. Each manufacturer will be able to choose the procedure(s) that best fits their processes.
|Table 2. Proposed List of Elements and Limits5|
|Element||Parenteral or Inhalational Daily Dose (µg/day)||Oral, Topicals, and Dermal, Mucosal Daily Dose (µg/day)|
(Combination not to exceed)
(Combination not to exceed)
|The above limits are derived from conservative calculations based on 50Kg (110lb) body weight and 10g daily dose, assuming a 70-year life span. Bioavailability assumptions: oral 10%, parenteral 100%. Compliance options may be demonstrated by analysis of the drug product at maximum daily dose and compared to limit level (modified daily dose permitted daily exposure) or summation of the impurity level in each of the components of the drug product.4,5|
In moving from a chemical to an instrument based methodology, the USP has taken great care to allow for a flexible approach and is working closely with both the FDA and industry to ensure widespread agreement on interpretation of the revisions. Following are brief descriptions of the methodologies being proposed.
Sample preparations range from relatively simple acidification and direct injection to more complex total oxidations/dissolutions performed under elevated temperature and pressure in appropriate acid(s) to assure dissolution of target elements. Sample preparations are intended to yield an aqueous digestate suitable for instrumental analysis via one or more instrumental techniques.4,7 (See Figure 1 for a decision tree on sample preparation and analysis.)
Figure 1. Sample Preparation Decision Tree
The techniques typically utilized for the analysis of the sample digestates are Cold Vapor Atomic Absorption (CVAA), Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP/OES), and/or Inductively Coupled Plasma-Mass Spectrometry (ICP/MS). Technical considerations beyond the scope of this discussion dictate the choice of procedures. As with any analytical technique, interferences (chemical and/or physical) exist with each technique. Intelligent decisions relative to the elements of interest and the sample matrix will indicate the appropriate analytical approach.
Although the majority of applications can be satisfied by the use of ICP/MS and/or ICP/OES, expert trace metals chemists recognize that alternative procedures are required at times to satisfy unusual analytical challenges. Careful examination of each application must be done from a quality assurance perspective. There are situations when multi-element techniques that utilize the plasma as an ion source or light emission source are capable of producing values that appear to be valid from a quality control standpoint, but are nonetheless invalid from a quality assurance standpoint. On these occasions, the following instrumental techniques still play a role in a fully functional trace metals laboratory: Purge & Trap Cold Vapor Atomic Fluorescence Spectroscopy (P&T-CVAFS), Graphite Furnace Atomic Absorption (GFAA), Flame Atomic Absorption (FLAA), and Gaseous Hydride Atomic Absorption (GHAA).
The revised quantitative methods, though of great benefit in terms of accuracy and recovery, are significantly more expensive than the qualitative USP<231>. Perhaps the main criticism of the revised testing protocols relates to the associated cost of new instrumentation and/or outsourcing for testing. Because atomic spectroscopy and ICP spectrometry are not yet widely used in the pharmaceutical industry, smaller manufacturers and excipient companies may not yet have the instrumentation in place and will need to either purchase the new equipment or send their testing to contract laboratories.
The various instrumental techniques each include advantages and disadvantages with respect to cost, sensitivity, selectivity, and ease of use. Some of the techniques are best suited for certain elements, but not for others. The same is true for certain sample matrices. For example, the analysis for lead and arsenic by ICP/OES or FLAA frequently represents a poor choice because of the associated high detection limits (DL). With these elements sample preparation would have to be more complicated in order to offset relatively high DL for the instrumentation. Alternatively, GFAA or ICP/MS would be preferable choices.
The instruments listed in Table 3 are capable of performing analysis of some or all of the elements listed in USP<232>. This table compares the instruments and equipment most commonly required to meet the USP requirements. Approximate values representing initial purchase and ongoing operating costs as well asl abbreviated summaries of strengths and weakness are also listed.
|Table 3. Instrumentation|
|Instrument/ Equipment||1. Purchase Price
2. Operating Costs (annual)
|Graphite Furnace Atomic Absorption (GFAA)||1. $30,000 – $65,000
|Sensitive and selective; good for metalloids that suffer poor ionization and are weak light emitters.||Low detection limits and good selectivity when Zeeman BG used; proper temperature programming overcomes abbreviated digestions.||Single element technique; consumables are costly; higher skill level to operate.|
|Flame Atomic Absorption (FLAA)||1. $15,000 – $40,000
|Commonly used for alkali metals.||Easy and relatively inexpensive to operate; accurate and sensitive for alkali metals.||Single element technique; not sensitive for heavy metals; subject to uncorrectable interference.|
|Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP/OES)||1. $50,000 – $100,000
|Excellent multi-element technique with relatively good sensitivity and selectivity when configured correctly.||Rapid multi-element analysis produces relatively low detection limits; excellent for alkali and alkaline earth elements; large linear dynamic range; tolerance to high levels of dissolved solids; axial and radial viewing of the plasma provides high versatility; essential backup for situations where uncorrectable interferences exist for ICP/MS.||Occasionally stymied by uncorrectable spectral overlap; elements of significance to USP (As, Pb, Hg) are not sensitive enough for many applications.|
|Inductively Coupled Plasma Mass Spectrometry (ICP/MS)||1. $130,000 – $180,000
|Multi-element ultra trace technique.||Superior sensitivity; selectivity excellent when configured correctly and applications investigated thoroughly; excellent for high mass elements; many polyatomic interferences can be removed via collision or reaction cell technology; rapid determinations possible.||Higher skill level to operate; initial and ongoing cost is high; occasionally stymied by uncorrectable isobaric interference.|
|Digestion||1. $500 for microwave digestion bomb; $40,000 for microwave system; $35 for oven digestion bomb;
$4,000 for convection oven.
|Use dependent on the matrix under test. Note that essentially equivalent, efficient and less expensive alternatives are available rather than dedicated systems. Variations in acid matrix and heating times fluctuate with material being digested. Near complete oxidation of organic carbon to CO2 and water is important when ICP/MS is used to avoid enhanced ionization of certain elements and/or carbon-containing polyatomics.|
Validation of Quantitative Procedures
Verification of the compendial procedures indicated in USP<233> will be required prior to use. This can be completed by meeting the “Procedure Validation Requirements” outlined in USP<233>.4 Two types of validations (limit and quantitative) will be permitted. The limit test validation will include limit of detection, precision, and specificity. The quantitative test validation will include performing accuracy, precision, specificity, limit of quantitation, range and linearity. Both types of validations will need to be verified experimentally. In addition, sample preparation not specified in the monograph will also require verification. The compendial procedures encompass both ICP/OES and ICP/MS technologies, and the general instrumental and suitability requirements for each procedure are specified for users. Laboratories will be able to choose the appropriate technology that best fits their needs.
Although USP<233> represents a major shift for the pharmaceutical industry, U.S. Pharmacopeia has clearly stated they do not intend to create a system of unnecessary and complicated requirements.5 The goal is simply to create standards for safer pharmaceutical products and dietary supplements, through the use of modern technology. While increased cost is a factor, and manufacturers will need to make certain adjustments, this shift represents an appropriate modernization that manifests itself by assuring higher quality products.
- Raghu ram, P. “IPC-USP 75th Annual Scientific Meeting 2008.” USPOrg. 2008. Hetero. http://www.usp.org/pdf/EN/meetings/asMeetingIndia/2008Session4track2.pdf. Last accessed Januray 20 2011.
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- Lira, Sergio, Peter Brush, Laurence Senak, Chi-san Wu, Edward Malawer. “The Use of Inductively Coupled Plasma-Optical Emission Spectroscopy in the Determination of Heavy Metals in Crospovidone and Povidone as a Replacement for the Concomitant Visual Comparison Test.” Pharmacopeial Forum Vol. 34(6)[Nov.-Dec. 2008].
- US Pharmacopeia Hot Topics Elemental Impurities Revised October 6, 2010. http://www.usp.org/hottopics/metals.html. Last accessed January 25, 2011.
- Elemental Impurities. Pharmacopeial Forum Vol. 36(1)[Jan.- Feb. 2010]
- Lenntech, Home Page, http://www.lenntech.com/periodic/periodic-chart.htm, January 19, 2011
- Standard methods for the examination of water and wastewater. 20th ed. American. Public Health Association, Washington, D.C. 4. Horwitz, W. (ed.) 2000.
- Stellmack, Mary and Dr. Kent Rhodes. “Metal Contamination in Biopharmaceutical Drugs: Solving a puzzle without all the pieces.” The McCrone Group. October 1, 2010. McCrone Associates Inc. http://www.mccrone.com/media/2010/10/14. Last accessed January 19, 2011.
- Taylor, Howard E. Inductively Coupled Plasma-Mass Spectrometry, Practices and Techniques. San Diego: Academic Press, 2001.
- Pedersen, Ole. Pharmaceutical Chemical Analysis: Methods for Identification and Limit Tests. Boca Raton: CRC, 2006.
- Wilbur, Steve. “A Comparison of the Relative Cost and Productivity of Traditional Metals Analysis Techniques Versus ICP-MS in High Throughput Commercial Laboratories.” Agilent. 2005. Agilent Technologies. http://www.chem.agilent.com/Library/applications/5989-1585EN.pdf. Last accessed January 20, 2011.
- Agency for Toxic Substances and Disease Registry (ATSDR). ATSDR.CDC.Gov. http://www.atsdr.cdc.gov/ToxProfiles/index.asp. Last accessed January 20, 2011.
- Electronic Code of Federal Regulations (e-CRF). http://ecfr.gpoaccess.gov/cgi/t/text/text-idx?c=ecfr&tpl=%2Findex.tpl. Last accessed January 20, 2011.
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