A Novel Sample Preparation Method for Complete Digestion of Complex Pharmaceutical Matrices

For more than 100 years, the USP General Chapter <231> concomitant visual test was the standard method for the determination and quantification of heavy metal impurities present in pharmaceutical products and raw materials. Based in principle upon the precipitation of insoluble metal sulfides, General Chapter <231> sample preparation involves ashing the sample at 600–800 °C followed by acid digestion of the resultant residues. Improvements in analytical equipment and techniques, however, have elucidated several known drawbacks of this technique. Considerable amounts of known volatile elements (As, Hg, etc.) may be lost during the ashing step, and the inability of some elements (As, Cd, Hg, etc.) to form colored complexes with sulfide ions can lead to under-recoveries of these heavy metal impurities. (Moreover, the time required for the ashing and digestion procedure can be in excess of 4 hr, slowing the acceptance of raw materials and shipment of finished products.) As a result of these frailties, USP recently approved General Chapters <232> and <233> to replace General Chapter <231>.1 Compliance with these two new chapters is required by May 1, 2014.

A brief summary of General Chapters <232> and <233>

Chapter <232> provides concentration limits of a number of metal impurities that may be present in pharmaceutical products and raw materials:

  • Elements of Class 1: As, Hg, Cd, and Pb (the so-called “Big Four”) are known, or are strongly suspected to be, human toxicants and should be essentially absent (<2.5 μg/g for drug substances and excipients)
  • Elements of Class 2: Cr, Cu, Mo, Ni, V, Pd, Pt, Os, Rh, Ru, and Ir are catalyst- and envirocontaminants that are comparatively less toxic, but can affect the stability and shelf-life of products due to their catalytic nature.

Chapter <233> describes sample preparation procedures that can be employed prior to inductively coupled plasma (ICP) analysis. Dissolution of the sample in an aqueous or organic medium and closed-vessel (microwave) digestion are discussed in the chapter.

Challenges of acid digestion

Although the closed-vessel sample preparation techniques proposed in Chapter <233> offer improved accuracy, sensitivity, and elemental specificity, the question of the broad applicability over a wide range of organic compounds used in the pharmaceutical industry remains. For example, many modern drugs have enteric coatings that consist of complex synthetic polymers or biopolymers. Using even the most aggressive acid mixtures in high-temperature and high-pressure environments, complete digestion of these complex components is not certain. Consequences of incomplete digestions include high residual carbon content; metal losses due to complexation; and high blank values, all of which can negatively impact the inductively coupled plasma-optical emission spectrometry/mass spectrometry (ICP-OES/MS) analysis step.

Recently, a feasibility study with a variety of over-the-counter (OTC) aspirin samples was conducted by Evans Analytical Group (Liverpool, NY).2 The aim was to show efficacy of the combined microwave-induced oxygen combustion technique coupled with ICP-MS analysis for pharmaceutical products.


Four OTC aspirins with the same active pharmaceutical ingredient (API)—acetylsalicylic acid—were examined. Thermogravimetric analyses revealed different mass loss patterns indicating different coating, buffering ingredients, or formulation excipients. The major matrix component present in aspirin samples—carbon— ranged from 30 wt% to 49 wt%.


Figure 1 – Multiwave PRO microwave reaction system.

The Multiwave PRO microwave reaction system (Anton Paar, Ashland, VA) (Figure 1) equipped with the Rotor 8NXQ80 was used for the sample preparation. The rotor is supplied with eight quartz glass vessels and has simultaneous pressure measurement capability on all digestion vessels. Simultaneous pressure measurement enables the rotor to proactively detect exothermic reactions and take suitable action (such as reducing the microwave power) to keep it within safe temperature and pressure limits. Since the rotor has the ability to reach maximum reaction conditions of 300 °C and 80 bar, it is suitable for use with the most challenging samples.

Due to the enteric coatings on the aspirin samples, the microwave-induced oxygen combustion procedure was also used. This procedure combines the advantages of an ashing/combustion technique with closed-vessel acid digestion in a single preparation step. The method prevents analyte losses and matrix effects, while eliminating the need for concentrated acids or solvents that may interfere with the analysis step.

Figure 2 – MIC vessel cross-section: Simultaneous ashing/combustion and acid digestion.

Microwave-induced oxygen combustion was performed in the quartz vessels of the Rotor 8N configuration used with the Multiwave PRO microwave reaction system. A PerkinElmer (Waltham, MA) ELAN DRC II ICP-MS system was used to measure the concentrations of the selected elements. As internal standards, 45Sc, 89Y, 115In, and 159Tb were used.

Analytical procedure

Whole tablets ranging in mass from ca. 400 to 700 mg were placed into the sample pellet holder and the microwave-induced oxygen combustion (MIC)/digestion procedure was performed. Three replicates of each tablet were repeated.

The sample holders were mounted with samples and positioned in the quartz digestion vessels (Figure 2). Six milliliters of 20% (v/v) diluted HNO3 solution was pipetted into each quartz vessel, closed, and loaded into the Rotor 8N. After placing the rotor into the Multiwave PRO microwave reaction system, the vessels were pressurized with 20 bar of high-purity oxygen. With microwave irradiation applied, the samples were ignited, reaching temperatures of approx. 1000 °C. This MIC application enabled total and complete mineralization. Digestion blanks and calibration verification standards were processed with every MIC digestion.

The microwave method had a total processing time of approx. 45 min (including heating and cooling). The digestion solutions were all clear, with a residual carbon content of <1%, highlighting the efficacy of the combustion/digestion technique.

Table 1 – Validation of the digestion efficiency of the MIC procedure*

Evaluation: MIC procedure

The microwave-induced oxygen combustion method was evaluated by spiking the absorbing solution prior to digestion with each element in a magnitude equal to 1 and 10 μg/g for the solid samples. Any losses of elements during the digestion would be critical for evaluation and validation of the digestion method against the conventional USP procedures. Table 1 shows the recovery data of the MIC procedure in % for Aspirin A.

The four Class 1 elements (As, Cd, Hg, and Pb) marked in gray showed recoveries between 96% and 102% for both spike levels. The recoveries for the Class 2 elements, with the exception of osmium, ranged from 86% to 112%. The high value of osmium can be explained as the formation of volatile osmium tetroxide (in the oxidative nitric acid digestion medium) causing false high readings. A longer nebulization time, or the use of hydrochloric acid for osmium analysis, eliminates these false high readings.

Evaluation: ICP-MS procedure

Incomplete digestion of samples can lead to matrix effects, reducing the accuracy of measurement methods. To examine matrix effects, sample solutions were spiked postdigestion at 10- and 100-μg/L levels representing concentrations in the tablets at 1 and 10 μg/g.

The results in Table 2 demonstrate excellent recoveries for almost all elements of interest, at both low and high spike levels. This suggests a minimized matrix effect due to near-complete mineralization during the combustion and digestion procedure.

Table 2 – (Confirmation of) complete sample mineralization via MIC procedure*

Detection limits: MIC-ICP-MS procedure

The detection limits (DL) of the MIC-ICP-MS method are shown in Table 3 (calculated from 3σ of the digestion blank; n = 10).

Table 3 – Detection limits of the MIC-ICP-MS method for USP <232> elements*


Upon evaluation from independent parties, closed-vessel microwave-induced oxygen combustion combined with ICP-MS measurement (MIC-ICP-MS) is proven as an effective assay of elemental impurities in pharmaceutical products, as demonstrated on enteric-coated aspirin samples of various brands. Complete decomposition of organic matrices—independent of their structure and functional groups—resulted in superior recovery data and detection limits when compared to legacy preparation techniques and other closed-vessel digestion techniques.

The coupled MIC-ICP-MS technique provided spike recovery data between 86% and 112% for the Class 1 and Class 2 elements, exceeding the requirements proposed in USP <232> (recovery between 80% and 150%). Detection limits also meet the USP <232> criteria.

The Multiwave PRO microwave reaction system is a powerful tool to assist with full compliance to USP <232> and <233> requirements, offering:

  • Closed-vessel digestion
  • Simultaneous high-temperature and high-pressure capabilities that ensure total digestion of all sample types
  • Special applications such as microwave-induced oxygen combustion that enable total mineralization of even the most challenging sample matrices, independent of structure and chemical composition.

To further assist pharmaceutical companies preparing for USP Chapters <232> and <232>, Anton Paar also offers the Pharma Qualification Package (PQP), an extensive instrument Installation Qualification documentation folder. The PQP complies with pharmaceutical regulations such as GMP, 21 CFR Part 11, GAMP-5, and USP Chapter <1058>, and follows the 4Q model—DQ, IQ, OQ, and PQ (design qualification, installation qualification, operational qualification, and performance qualification). The PQP documentation package for the Multiwave PRO contains a traceability list, risk analysis, user Standard Operating Procedures (SOPs), as well as a protocol for 21 CFR Part 11 compliance (related to software functionalities such as audit trail and e-signature).


  1. U.S. Pharmacopeial Convention; http://www.usp.org/usp-nf/official-text/revisionbulletins/elemental-impurities-limits-andelemental- impurities-procedures-0.
  2. Nam, K.H.; Isensee, R. et al. Spectroscopy2011, 26(4), 2–7.

Reynhardt Klopper is Product Specialist, and Eric Fox is National Sales Manager, Analytical and Synthetic Chemistry, Anton Paar USA, Inc., 10215 Timber Ridge Dr., Ashland, VA 23005, U.S.A.; tel.: 804-550-1051, ext. 135; fax: 804-550-1057; e-mail: eric.fox@anton-paar.com.