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ppm Detection Limits with mg Sized Samples

USP <231> Heavy Metals

The limit test for heavy metals is a qualitative test that demonstrates that the content of metallic impurities that are colored by sulfide ion does not exceed the specified limit.  Metals that typically respond to this test are lead, mercury, bismuth, arsenic, antimony, tin, cadmium, silver, copper, and molybdenum.

There are three different sample preparation techniques that can be utilized in this analysis. Method I, II, or III may be specified in individual monographs. Method I is used for substances that produce a clear colorless solution. After addition of a sulfide reagent, the color is compared to both a standard as well as a sample spiked at the limit. In Method II the substance is first carbonized by heating with sulfuric acid, then the carbon is burned in a muffle furnace. Metals are then extracted from the residue, and the analysis can proceed free of any organic interference. Method III is a sulfuric and nitric acid digestion method followed by oxidation with peroxide.

The Problem: Sample Size

Both USP and FDA acknowledge problems with this test both in terms of the specificity and lack of quantification, even for monograph materils.

In addition, USP<231> requires a large sample.  Sample size for USP<231> is determined by the following formula: 2.0/(1000L), where L is the limit in percentage. Thus for a limit of 0.001% (or 10 ppm), at least 2 g of material is required. This may be impractical for many substances such as proteins or peptides. For this reason, we offer a more sensitive and specific test using ICPMS which requires as little as 10 mg samples. 

ICP-MS (Inductively Coupled Plasma-Mass Spectrometry)

While USP<231> does not give a specific result for any of the elements, ICP-MS can identify and quantify each metallic impurity with higher sensitivity. ICP-MS can be used to test for just the heavy metals or practically the whole Periodic Table in one analysis (>60 elements). USP<730> now covers ICP-MS.

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ICP-MS, An Alternative

Alternatives to USP<231> include some of the spectroscopic techniques such as GFAA, ICP-OES, and ICP-MS.  Of these, ICP-MS is the method of choice.

ICP-MS

  • is the most sensitive technique, having the lowest detection limits (0.01-1 ug/L in solution), 
  • is a fast, multi-element technique (up to 60 elements in a 2 minute scan)
  • has definitive, multiple isotope identification (less prone to interferences)

Typical detection limits for ICPMS are 0.01-1 ug/L (ppb) in solution. Sample preparation usually consists of simply diluting the sample in 1% nitric  acid although we offer a variety of digestion techniques.  Since ICP-MS is the most sensitive technique for trace elements in solution, a 10 mg sample will give detection limits for most elements in the range of 0.01-1 ug/g (ppm). More than 60 elements can be determined in the same analysis which is a big advantage over GFAA where 

each  element is done separately. We offer a variety of single element, multi-element including just the <231> heavy metals, and complete (>60 element) metals screens.

ICPMS Plasma

Some proteins bind metals, and the metal assay is important for QC. Here again, using only very small samples, ICP-MS can accurately assay the metal content. 

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For more information on ICP-MS

ICP-MS, the technique

ICP-MS Metals Screen

Pb by isotope dilution-ICP-MS

Arsenic Speciation by IC-ICP-MS

ICP-MS Cell Technology


Elemental Impurities
Jan 2010

In the January/February issue of Pharmacopeial Forum, USP has proposed two new general chapters: Elemental Impurities - Limits <232> and Elemental Impurities - Procedures <233> [PF 36(1), Jan-Feb 2010]. These chapters are proposed to replace Heavy Metals <231>. In addition, USP also proposed a new dietary supplement general chapter: Elemental Contaminants in Dietary Supplements <2232>.  The intent is to replace an outdated, non-specific method <231> with a more modern, specific method. While the details of how these methods should be implemented are uncertain, this article summarizes the current intent of USP to modernize the control of inorganic impurities and harmonization with ICH Q3A(R2): Impurities in Drug Substances and ICH Q3B (R2): Impurities in New Drug Products.

USP Elemental Impurities,
Hot Topics

USP Stimuli Article, Dec 2008

ICH Q3AR, Impurities

ICH Q3A(R2), New Drug Substances

ICH Q3B(R2), New Drug Products


USP<232> Limits

In <232>, elements are divided into two categories. Class 1 consists of highly toxic elements (The "Big Four"): arsenic (As), cadmium (Cd), lead (Pb), and mercury (Hg). Class 2 impurities include catalysts which may have been introduced during processing such as chromium (Cr), copper (Cu), manganese (Mn), molybdenum (Mo), nickel (Ni), palladium (Pd), platinum (Pt), vanadium (V), osmium (Os), rhodium (Rh), ruthenium (Ru), and iridium (Ir). Limits for each element are based upon EMEA guidelines.

Testing for Class 1 is mandatory, but testing for Class 2 elements is mandatory only for those elements which may be added during manufacturing or those expected to be present in the raw materials. Pass/Fail is determined based upon Permissible Daily Exposure (PDE), calculated either from the analysis of the drug product as a whole or from a summation of the determination of inorganic impurities in the individual components.

USP<233> Procedures

A decision tree is presented in <233> to select an approach for the analysis of heavy metals. Unless the solid sample is to be tested directly, using analytical techniques such as X-Ray Fluorescence (XRF) or Laser Ablation-ICPMS (LA-ICPMS), then solubility in aqueous media should be assessed. If the sample is soluble in aqueous media, then the sample is diluted or dissolved in an appropriate aqueous solution and analyzed. We presume this aqueous media includes a simple acid dissolution or digestion procedure. If the sample is not soluble in aqueous media, then solubility in organic solvents should be assessed. If the sample is neither soluble in aqueous media nor organic solvents, then the sample should be subjected to the referee method consisting of closed vessel microwave digestion and either ICP-OES or ICP-MS analysis.

As always, methods must be validated, and <233> proposes steps for the validation of both limit and quantitative tests, the latter approach being recommended.Either ICP-OES or ICP-MS may be used to demonstrate compliance with USP <233>. However, in our experience, the limits proposed in <232> makes using ICP-OES inadvisable because of insufficient sensitivity of this instrument. Dilution factors during sample preparation can range from 100-1000, which means that elements with limits near 1 g/g are present in the solution at 1-10 g/L. This range in solution is near the limit of detection (LOD) for most elements by ICP-OES, while LODs using ICP-MS are generally 0.1 g/L or less. This makes ICP-MS a much more suitable instrument for this analysis. For some materials, dilution factors of <100 are possible, and ICP-OES could meet the sensitivity needs of this analysis..

 


Method Validation

Method validation requirements for <233> performed as a quantitative test include demonstrating the following parameters: accuracy over 50-150% of the limit, precision (repeatability and intermediate precision), and specificity. Limit of Quantitation (LOQ) is only required to be demonstrated as acceptable accuracy near the maximum limit. Notably lacking from this list of requirements is robustness and the stability of the sample solution.

Issues and Concerns

While <233> does not mention <730>, presumably the requirements of the Plasma Spectroscopy General Chapter will be implemented.

System suitability, or control limits for Drift, for ICP-MS is not well defined in either <233> or <730>. The agreement between continuing calibration standards in <233> (NMT 20%) is not in agreement with <730> (NMT 10%).

System suitability for ICP-OES consists of the analysis of a check standard at 1 mg/L. However, sample limits may be 100 times lower, in the 10 ug/L range.

The analysis of calibration standards to demonstrate range and linearity are across a narrow range than those typically used by our laboratory. One of the benefits of ICP-MS instrument is its linearity of response across a very wide range.. We hope that our wider range muti-point calibration will satisfy the requirement.

 


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