ICP-MS Calibration and Detection Limit
ICP-MS was calibrated using a series of working standard solutions freshly prepared before sample analysis. The working standard solutions span a concentration range from 0.5 J to 2 J as defined by USP 233. Calibration is deemed successful when the coefficient of determination (R
2) of the calibration line is not less than 0.99.
A generic ICP-MS method was developed and validated for pharmaceutical analysis. The same method was also used in this clean chemistry study. The capacity of this generic method in detecting trace contaminants is reflected by method detection limit (MDL). MDL is defined here as ten times of the instrument detection limit (IDL). IDL is three times of the standard deviation of six replicate measurements of the calibration blank (i.e., the diluent used to prepare J solutions). Since MDL changes with instrument condition such as interface cone deposition and pump tubing wear, there is a slight fluctuation in the day-to-day MDL values. The averages of MDLs obtained during this study are shown in Table II. MDL values provide guidance to the capability of the generic ICP-MS method in detecting trace level contaminants. They were not verified experimentally. The method reports “n.d.” (not detectable) in the tabulated results if the measurement is below MDL. The concentrations are reported “as it is” in graphs for illustration purpose where negative concentrations are generally treated as n.d. For most target elements, this method achieves sub-parts per trillion detection limits. The high MDL of molybdenum most likely results from the high residual in concentrated acids.
The importance of water quality in trace elemental analysis cannot be overemphasized. For elemental impurities compliance, DI water quality meeting ASTM Type I requirement is considered sufficient. DI water needs to be monitored constantly for its resistivity and total organic carbon (TOC) level, with acceptance criteria resistivity not less than 18 megohm cm at 25°C and TOC not more than 10 ppb. For information purpose, we measured 10 independent DI water samples and compared their elemental impurities to 10 independent tap water samples in Table III. Most target elements are not detectable in DI water. Copper has a concentration 2.088 μg/l. However, this concentration is considered insignificant as compared to the copper concentration 300 μg/l at 1 J level (Note: 1 J is defined here based on a dilution factor of 1000, i.e., 1 mg sample dissolved in 1 ml diluted acid solution.).
Contamination from Sampling
Sampling is defined here as presenting an appropriate amount of sample representative of the pharmaceutical material under study for sample preparation. Once a sample is received, the analyst should take caution not to contaminate the sample when opening the package, dispensing, homogenizing, and weighing the sample. In most cases of solid dosage form sampling, the unit dose far exceeds the capability of microwave digestion and ICP-MS instrument and weighing a homogeneous sample in the form of powder is required. A variety of apparatus exist for pulverizing and homogenizing tablets and capsules, including mortar and freezer mill. Freezer mill is able to homogenize capsule products. To evaluate contamination from sample pulverization, a tablet product with a nominal unit weight of 138 mg was ground into fine powder using two different methods: glass mortar and freezer mill. For each pulverization method, 10 powder samples each of 138 mg were prepared using a generic approach applicable to most pharmaceutical materials with excellent method robustness: The powder sample was solutionized completely in a mixture of 5 ml concentrated HNO3 and 1 ml concentrated HCl by closed-vessel microwave digestion; Microwave digestion was performed at 200°C with a holding time of 5 min; And digest was transferred into 100-ml PMP flask and diluted with water. An aliquot was then transferred into a 14-ml polypropylene test tube for ICP-MS analysis. For comparison, 10 intact whole tablets were also prepared and analyzed for the target elements using the same generic method.
Side-by-side comparison of these three different sampling methods can be seen in Fig. 1. Contamination from sample pulverization is low even for copper and nickel. There is no statistically significant difference in these three sampling methods with regard to the level of contamination.
Contamination from Microwave Apparatus
Pyrex glass vessel can be used for closed-vessel microwave digestion as long as HF acid is not used and rigorous cleaning procedure is followed. The cleanliness of digestion vessel, stirring bar, and vessel cap was subject to extra scrutiny because of the readiness of contaminants leaching into concentrated acid mixture under high temperature and pressure. We have identified Teflon-coated stirring bar as the culprit of cobalt contamination. Glass-encapsulated stirring bar should be used with microwave digestion whenever HF acid is not required for total extraction.
The microwave system uses Teflon-lined plastic cap to seal and vent the digestion vessel during closed-vessel digestion. To evaluate cap cleanliness, we soaked 50 caps overnight in two different extraction media: 500 ml DI water and 500 ml 10% nitric acid. The solutions were then analyzed for impurities. The concentrations of the target elements found in extraction solutions are compared to those found in the control samples which are 10% nitric acid stored in pre-cleaned PMP flask. The comparison is shown in Fig. 2. Given the significantly high concentration of nickel, copper, and lead extractables from caps in 10% nitric acid, it is suggested that the caps be soaked in 10% nitric acid overnight and rinse with DI water at least three times before use with digestion vessel for microwave digestion.
Contamination from Pyrex digestion vessel was also evaluated by an extractables study. We filled 10 new 35-ml digestion vessels with 30 ml 10% nitric acid and let the vessels sit on bench overnight. Target elements in the extraction media were analyzed and the results are shown in Fig. 3 along with the results from the control samples. The control samples are 10% nitric acid stored in a PMP flask. Copper, arsenic, and lead show a significantly higher concentration in the extraction media as compared to the controls, which indicates the potential contamination with these elements from digestion vessel. It is recommended that the Pyrex digestion vessels be soaked in diluted nitric acid overnight and then be rinsed with DI water before their use for microwave digestion. Method blanks show that contamination from repeated use of Pyrex vessels is insignificant for the USP 232 target elements. Pyrex glass vessel, without the Teflon liner, can be used for acid digestion as long as the vessel is properly cleaned prior to use. It is contrary to the common perception of “dirty” glass digestion vessels as compared to the “cleaner” quartz or Teflon vessels.
Contamination from Plastic Container
Plastic containers made from various materials such as polyethylene (PE), perfluoroalkoxy (PFA), polymethylpentene (PMP), and polypropylene (PP) are widely used in elemental analysis labs for sampling and storage because of their cleanliness and compatibility with HF acid. Contamination from plastic container, although less severe than other container types, is worthy of study (5). We tested the cleanliness of 14-ml PP test tubes used with ICP-MS auto-sampler. Before being filled with diluent, the test tubes were cleaned in two different ways: DI water rinsing and soaking in 10% nitric acid overnight and then DI water rinsing. Target elements found in diluent are shown in Fig. 4, along with the elements found in diluent filled directly into test tubes that were not treated. There is nickel contamination from the un-treated test tubes, but the contamination level is significantly reduced by tube cleaning. It is interesting to see that the acid soaking followed by DI water rinsing brings about the same cleanliness as DI water rinsing alone. It seems that DI water rinsing is sufficient for PP test tube cleaning. Similar results were also observed for 50-ml PP conical sample tubes and are not shown here. PMP flasks and PFA beakers were not studied for their cleaning, but method blanks have not shown any significant contamination when we used these plastic containers and employed a DI water rinsing cleaning procedure for them. For USP 232 compliance purpose, contamination from plastic containers is considered low and does not impact method capacity. However, it does not exclude acid soaking as an ultimate cleaning method when lower detection limit is needed. For new containers, plastic or glass, acid soaking followed by thorough DI water rinsing is always recommended before their first use.
Contamination from Lab Supplies
Any lab supply, such as gloves, wipes, Parafilm, pipette tips, and transfer pipettes, can be a potential source of element contamination if they come in indirect or direct contact with sample. We evaluated two brands of powder-free nitrile gloves used in our labs: small-size Kimberly-Clark exam gloves and small-size High Five gloves. One glove was dipped and rinsed several times in 100 ml 10% nitric acid in a PFA beaker. Rinsates were analyzed for the target elements and the results are shown in Fig. 5 along with the element concentrations in the control samples. Control samples are 10% nitric acid stored in a pre-cleaned PFA beaker. The High Five gloves exhibit very high level of surface contamination especially with lead. Glove surface was also contaminated with arsenic and cadmium. By contrast, Kimberly-Clark gloves show significantly less contamination. Cleaner clean room gloves are commercially available for elemental analysis. In any case, an analyst should use DI water to thoroughly rinse their gloves before working in the lab.
Contamination from Parafilm, Kimwipes, and Scott towels was also evaluated in an extractable study because they came in direct or indirect contact with samples. One 10 cm × 30 cm Parafilm was soaked in 50 ml 10% nitric acid for 30 min. One ply of 11 cm × 21 cm Kimwipes was soaked in 50 ml 10% nitric acid for 30 min. One ply of Scott c-folded paper towel was soaked in 50 ml 10% nitric acid for 30 min. All extraction media were then sampled and analyzed for target elements. Figure 6 shows the concentrations of target elements in the extraction media for each material. Control sample is 10% nitric acid stored in a PFA beaker. Scott paper towel shows very high level of vanadium, cobalt, nickel, copper, and lead in its extractables. Although these extractables are not readily going to samples, Scott paper towel is considered dirty and presents a potential contamination source. Kimwipes is much cleaner, but still shows a high concentration of nickel and copper in its extractables. Its direct contact with sample should be avoided. Thus, using Kimwipes for cleaning and drying should be limited, if cannot be avoided, in elemental analysis lab. Air drying is preferred for wet containers. None of the target elements is detectable in Parafilm extractables and control samples. Parafilm is considered clean and suitable for use in elemental analysis lab.
Contamination from Laboratory Atmosphere
Contamination from dust and airborne particles in an elemental analysis lab has long been identified (6). While a clean room facility is usually recommended by authorities for trace element analysis (7), the lab should always makes its decision by evaluating air contamination and its effect on lab operation through air monitoring or experiment. To evaluate the contribution of lab air to elemental impurities contamination, we placed two 500-ml PFA beakers side-by-side on the bench of sample preparation lab. Both beakers were filled with 100 ml 10% nitric acid. One beaker was open to air while the other was covered with Parafilm. After 5 days, the solutions from both beakers were aspired directly to ICP-MS and analyzed for target elements. The results are shown in Table IV in relative amounts. To correct for solution volume change caused by evaporation, all measured concentrations were normalized to osmium since osmium, if present, has extremely low concentration in air (8). As compared to the control which is the solution isolated from air, the solution exposed to air shows elevated elemental composition, especially for lead whose concentration is five times higher and iridium whose concentration is eight times higher. For laboratories located in polluted area or for laboratories having high dust level, lead contamination may become even more severe, which would require installation of a laminar flow hood or a clean bench. Clean bench supplies the work area with air cleaned by HEPA filter. HEPA filters are 99.97% efficient in removing particles down to 0.3 μm (9). In any case, exposure of the solution to air should be minimized by capping the containers or covering the containers with Parafilm.
Contamination from Analyst
Careless handling of standard and sample can cause serious contamination. Touching of the surface that may come into direct contact with solution can result in contamination with a number of elements including sodium, chlorine, calcium, and lead. A study of lead contamination from human fingers showed an average of 3.1 μg of lead in 15 ml 1 N nitric acid after soaking two fingers for 2 min (10). Cosmetics, hair dyes, and jewelry are known to cause contamination in trace element analysis (11). The analyst must be careful in handling standards. Direct pipetting from standard bottle should be avoided to minimize cross contamination. Standard solution must be mixed thoroughly when preparing concentrations at parts per billion or lower level. The analyst must be aware of the history of labwares and rigorous cleaning has to be performed before vessels exposed to high concentrations can be used for trace element analysis.