Lee Davidowski is with PerkinElmer Life and Analytical Sciences, Shelton, Connecticut.

Lee Davidowski

This article describes the use of an in-line, autodilution, and autocalibration sample delivery system coupled to an inductively coupled plasma–mass spectrometry system to analyze a group of over-the-counter pharmaceutical products. The study will follow the method protocol outlined in the recently approved 

 Chapters <232> and <233> on the determination of elemental impurities in the major four drug delivery systems.

The standard method for measuring elemental impurities in pharmaceutical products has been based on a 100-year-old "Heavy Metals Test," described in Chapter <231> of the 

) (1). This test involves precipitation of the metal sulfide in the sample, and compares the intensity and color of the precipitate to a lead standard. Even though this test has been used for more than 100 years, it is well-accepted that it is prone to error because it is not specific to any particular heavy metal, it suffers from quite severe matrix interferences, it is prone to very low recoveries, and it requires a skilled analyst to interpret the color correctly. For this reason, four years ago the USP organization set up an expert committee to investigate the viability of alternative analytical approaches to get around all these problems and replace Chapter <231> with a more robust and reliable instrumental technique.  

In April 2012, USP came out with a brand new inductively coupled plasma–optical emission spectrometry (ICP-OES) and inductively coupled plasma–mass spectrometry (ICP-MS) method to determine a group of metallic contaminants in pharmaceutical products. This method was summarized in General Chapter <232> entitled "Elemental Impurities – Limits" and Chapter <233> entitled "Elemental Impurities – Analytical Procedures,", which will be published in the 

 on December 1, 2012 (2). Pharmaceutical stakeholders will then have until May 2014 to implement these changes and be consistent with the new method described in Chapters <232> and <233>. 

This study focuses on the practical benefits of an ICP-MS system coupled to an in-line, autodilution, and autocalibration sample delivery system to determine a group of toxicologically relevant elements in various pharmaceutical products. It provides an overview of the 

 methodology with particular emphasis on the impurity levels and the recommended analytical procedure. It also analyzes some common pharmaceutical products covering the major drug delivery systems of oral, intravenous, and inhalational, and presents performance figures of merit for the system based on the 

Elemental Impurities in Pharmaceutical Products

Chapter <232> specifies the list of elements and their toxicity limits, defined as maximum daily doses of the different drug categories — oral, parenteral (intravenous injection), inhalation, and large-volume parenteral, whereas Chapter <233> deals with the sample preparation, analytical procedure, and quality control (QC) validation protocol for measuring the elements, including the choice of either inductively coupled plasma–atomic emission spectroscopy (ICP-AES), ICP-MS, or the use of an alternative technique as long as it is fully validated to be acceptable and equivalent to the plasma-based procedures described in the method.  

Chapter <232> Elemental Impurities — Limits

Table I shows the maximum permissible daily exposure (PDE) values (in micrograms per day) for the administration of drug products based on an "average" 50-kg person (3). The toxicity of an elemental impurity is related to its bioavailability. The extent of "chronic" exposure has been determined for each of the elemental impurities of interest for three routes of administration: oral, intravenous (parenteral), and inhalational. When the daily dose of an injection is >100 mL, the amount of element must be controlled through the individual components used to produce the drug product. This is known as the large-volume parenteral (LVP) component limit (in micrograms per gram) and is shown in the last column of Table I.  

Table I: Maximum permissible daily exposure (PDE) values (in Î¼g/day) for the administration of drug products based on an "average" 50-kg person 

This chapter also defines the maximum limit of the elemental impurities based on the material's final use and dosage. This is to ensure the drug manufacturers determine an acceptable level of impurity in the drug based on a typical maximum daily dosage of ≤10 g/day. These values are also used to take into account the sum of all the individual chemicals, active ingredients, and excipient compounds (fillers) used to produce the final drug product, which is intended to be the basis of discussions between the drug manufacturer and the suppliers of the raw materials.  

Chapter <233> Elemental Impurities — Analytical Procedure

This chapter describes the ICP-OES and ICP-MS analytical procedures (including sample preparation), together with the validation protocol for the determination of the elemental impurities in the drug products described earlier (4,5). 

To ensure the method is working correctly, a summary of the QC/QA validation protocols is given below. 

Target (element) limits (known as "J" values) defined as the acceptance value for the elemental impurity being evaluated, based on the weight, number of doses, and frequency of taking or administering the drug.

Calibration using two matrix-matched calibration standards and a matrix-matched blank. For each element, the high standard is twice (2J) the target limit and the low standard is half (0.5J) the target limit.

Samples are diluted so the concentration does not exceed 2× (2J) the target limits.

Samples are analyzed according to the manufacturer's suggestions for instrumental conditions and analyte masses, taking appropriate measures to correct for matrix-induced polyatomic interferences. 

A collision–reaction cell may be used to reduce the polyatomic spectral interferences.

A series of QC validation protocols, including spike recovery, accuracy, precision, and stability tests are followed to ensure the method is generating credible data. 

Sample preparation can be approached in four different ways. However, careful consideration should be given to the choice of acids because of the potential for matrix-induced spectral interferences generated in the ICP. 

Analyze neat, undiluted sample, if the sample is in suitable liquid form.

Dilute the sample in an acidified aqueous solution if the sample is soluble in water.

If the sample is not soluble in water, dilute it in an appropriate organic solvent. 

Use closed-vessel microwave acid digestion for insoluble samples. 

This study was performed using a NexION 300X ICP–MS system (PerkinElmer, Inc.) coupled with a prepFAST (Elemental Scientific Inc.) automated in-line autodilution and autocalibration system. The ICP-MS system has been described in the literature but is basically a single-channel version of PerkinElmer's Universal Cell Technology, which enables the instrument to be used in either the collision (KED) mode or the reaction (DRC) mode using one cell gas, in addition to the standard or normal ICP-MS mode (6).  

Figure 1: Schematic of the sample delivery system (prepFAST) illustrating a two-step process of loading sample into a loop and in-line dilution. During syringe reset in step 1, the loop is rapidly (vacuum) rinsed and loaded with the sample while the nebulizer and spray chamber are rinsed with carrier. In step 2, the sample is injected into the ICP-MS system along a flow path where in-valve addition and mixing of diluent (port 3) and internal standard (port 7) occurs.  

The autodilution system is fully integrated with the ICP-MS system for in-line autodilution of sample and stock standard solutions (7). Solutions are rapidly and reproducibly vacuum loaded from each autosampler location into a sample loop. This process is shown in Figure 1. Dilution, internal standard addition, and mixing all occur in line in the mixing valve. The system's high-resolution syringe-based delivery of all solution flow rates ensures precise and accurate dilution factors while maintaining a constant total sample flow rate. The system is used to autocalibrate the ICP-MS system and perform in-line dilutions of samples. Some of the benefits of this approach for the demands of a high-throughput pharmaceutical laboratory include  

Elimination of the errors associated with manual dilution steps

Reduction of reagent volumes required for high dilution factors

The samples investigated are shown in Table II, representing the three different categories of pharmaceutical products described earlier (oral, inhalation, and large volume parenteral).  

Table II: List of medications evaluated in this study 

For all medications, 1 g of sample was taken and brought into solution with an acid mixture of 2% HNO

 and 0.5% HCl. The arthritis pain medication was crushed into a powder and digested in a Multiwave 3000 microwave digestion system (PerkinElmer, Inc.) and made up to a final volume of 50 mL. The OTC cold and flu remedy was dissolved in the acid mixture, warmed on a hot plate, and made up to 50 mL. Both the allergy medication and the lactated Ringer's solution were already in liquid form, so they were prepared by diluting 1 g to a final volume of 50 mL. The oral medications were further diluted 10-fold in-line, using the autodilution system, to a final dilution factor of 500:1. It's worth emphasizing that if many different medications are being analyzed with large variations in daily dosage, the target values (J) change, requiring additional sample dilution or a new calibration graph. One of the major benefits of the in-line autodilution approach is that it can be done in a fully automated manner, without the need to make additional manual dilutions.  

Table III: ICP-MS instrumental conditions for the analysis of pharmaceutical compounds using USP methodology 

Depending on the target values (J) for the analyte of interest and the sample preparation technique used for the different drug compounds, 

defined calibration solutions at half (0.5J) and twice (2J) the target values are required. Table V (on-line) shows the PDE values (in micrograms per day) for the oral drugs and target concentration values (in micrograms per liter) calculated based on a daily dose of 10 g/day and a final sample dilution of 500:1 (10-fold in-line dilution of 1 g/50 mL). It can be seen that correlation coefficients for all calibration curves generated by in-line dilution (2% HNO

 and 0.5% HCl) of a single stock standard are 0.999 or better. 

Chapter <233> also defines a number of QC–QA protocols to validate the method, which include the following tests:  

Accuracy of the method by spiking the material under investigation at appropriate concentration levels related to the target limits and measuring the % recovery. 

Repeatability of the method by measuring six independent samples of the material under investigation, spiked at the target limits defined and measuring the recovery and precision of the measurements.

Articles

This article describes the use of an in-line, autodilution, and autocalibration sample delivery system coupled to an inductively coupled plasma–mass spectrometry system to analyze a group of over-the-counter pharmaceutical products.

ICP-MS with Autodilution and Autocalibration for Implementing the New USP Chapters on Elemental Impurities

Drinking water is essential for life and, in its most desirable form, is free of bacteria and chemical contaminants. Often, we assume that bottled water is cleaner than tap water, and in some parts of the world, that is certainly the case. Bottled water is popular in the U.S., outselling milk for the first time in 2006 (1). The largest consumer of bottled water (per capita) is Italy, hardly a developing nation (2). In the U.S., bottled water and drinking water contaminant regulations have been harmonized, so that both types of water should be of the same quality. However, in places with inadequate regulations or regulations that are not well enforced, bottled water can be more contaminated than expected.  

Water testing is critical to ensuring quality. Even though standards have been set and implemented to ensure the initial quality for a water, the bottling process can have unexpected consequences. For example, a famous bottled water was recalled after benzene was unexpectedly detected (3). A recent report found that 10% of bottled water in Shanghai, China, does not meet sanitary standards (4). Earlier this year, bottled water from Armenia was recalled because of contamination with more than 50 times the Food and Drug Administration–regulated amount of arsenic (5). Water is analyzed for three broad classes of contaminants: microbiological, organic, and inorganic. Inorganic contaminants include metals such as lead (Pb) and cadmium (Cd), that are clearly toxic. Some elements, such as selenium (Se), might be beneficial at low levels but toxic at higher concentrations. U.S.-regulated primary and secondary drinking water inorganic contaminant levels are shown in Table I.  

Table I: U.S. primary and secondary drinking water contaminants 

Bottled water from around the world has been collected and analyzed to compare the inorganic contaminants to those allowed in the U.S. Several analytical techniques are suitable for the measurements, and in this case, inductively coupled plasma-mass spectrometry (ICP-MS) was chosen for speed and detection level, using Environmental Protection Agency (EPA) method 200.8 (6). Detection limits 10 times below the concentration at which decision-making is required generally are recommended. Consensus methods and U.S. EPA methods generally require a number of quality control tests interspersed with samples for analysis to ensure high-quality data results for these important measurements. 

Table II shows a variety of bottled water results. The results are below the regulatory limit in all cases. For many elements, the concentration is below the detection limit as measured using the EPA definition. For several bottled water samples, it is interesting to note the presence of uranium. Although it is below the regulatory limit, it is clearly present and can be detected at very low levels by ICP-MS.  

Table II: Drinking water contaminants measured in a variety of bottled waters (Î¼g/L) using the PerkinElmer Sciex ELAN DRC II ICP-MS 

Disinfection with ozone rather than chlorine is becoming more popular for bottled water because it does not leave a residual smell or taste. Because bottled water is sealed after production, there is less chance that recontamination with bacteria will take place than in piped delivery systems. Because bromide (Br

) is present naturally in some water, ozone disinfection will promote the formation of bromate (BrO

), a carcinogen. Although total bromine can be measured with ICP-MS, it is not possible to tell how much of the bromine is present in the bromate form. Inorganic speciation is a growing area of analysis in which a separation of the various forms of the same element takes place before the elemental measurement. Several of the bottled waters reported earlier also were analyzed for bromate using ICP-MS coupled with high performance liquid chromatography (HPLC), which separated the Br

  before the metal determination. The U.S. and European regulated bromate level is 10 μg/L (ppb). 

Since the development of U.S. EPA method 321.8 in 1998, advances in speciation have taken place, allowing the method to be improved for ruggedness and speed. Figure 1 shows a chromatogram of 1 μg/L of bromate, showing that even at very low concentrations, it can be distinguished from the background levels (7).  

Figure 1: Chromatogram (isocratic LC method) of a standard containing 1 Î¼g/L (ppb) of bromate and bromide. Measured with the PerkinElmer Series 200 HPLC and ELAN DRC II ICP-MS, using Chromera software. 

Note that time for analysis is less than 3 min, whereas the method 321.8 chromatographic separation takes 8 min. Figure 2 shows overlaid chromatograms of a Chinese bottled water analyzed 49 times over the course of several hours to examine the short-term precision, which is better than 2%.  

Figure 2: Overlay of 49 chromatograms (isocratic LC method) of a Chinese bottled water sample obtained by consecutive injections. The average concentration and standard deviation for each species is shown. 

For a few samples with asymmetrical peaks and high bromate values, additional method development was done to ensure that the analyte of interest was separated adequately from any other components that might mistakenly contribute to the bromate measurement. A gradient HPLC method, although slightly longer than the isocratic method developed for routine use, showed that several peaks overlapped the bromate peak in some samples (8). Advanced interference correction on the ICP-MS system showed that the additional peaks did, in fact, contain bromine. Their identification was not investigated further. Figure 3 shows the chromatogram for bottled water purchased in Thailand showing the additional peak, uncovered with the gradient separation method. Table III shows the concentrations of bromide and bromate for a variety of bottled and tap water samples. The samples with suspect peaks or bromate concentrations above the regulatory limit were confirmed with the developed gradient HPLC separation to ensure that only one peak was present at the retention time expected for bromate.  

Figure 3: Chromatogram of a bottled water from Thailand with (a) an isocratic HPLC separation method and (b) a gradient HPLC separation method. 

Although few elements are regulated by their species, there are many whose form influences their toxicity. For example, chromium VI is known to be much more hazardous than chromium III. So in the future, as inorganic speciated analysis becomes more common, it is likely that measurements will yield more information on water toxicity than at present. Figure 4 shows chromatograms collected simultaneously for four elements and their species. Because ICP-MS is a rapid multielement detector, separation of several elements at once takes advantage of its multielement capability and provides a more efficient analysis (9).  

Table III: Quantitative determination of bromide and bromate in bottled and tap water samples confirmed with a gradient HPLC method (all units in Î¼g/L) 

Water analysis is critical to a safe and compliant bottled water supply. Measuring inorganic components is an important part of the complete analysis and can be performed efficiently using ICP-MS. Measurements should be performed on a regular basis to account for any changes that might have occurred at the source or in the bottling process.  

Figure 4: Simultaneous measurement of Br, As, Cr, and Se species with HPLCâICP-MS. 

Speciation measurements are a more recent type of analysis that couples two analytical techniques for the separation and detection of inorganic components. Speciation can be used right now for the determination of the disinfection byproduct bromate and might be further applied in the future as the species of other elements, such as arsenic or selenium, become regulated. 

 are with PerkinElmer Life and Analytical Sciences, Shelton, Connecticut. 

(1)  Water Industry News, May 4, 2007, http://

www.watertechonline.com/news.asp?N_ID=67248

(4)  Water Technology On-line, June 27, 2006. 

(5)  Water Technology On-line, March 27, 2007. 

(6)  R. Wolf, E. Denoyer, and Z. Grosser, EPA Method 200.8 for the Analysis of Drinking Waters, PerkinElmer Application Note D-6527, 2001. 

(7)  K.R. Neubauer, W.M. Reuter, P.A. Perrone, and Z.A. Grosser, Bromate/Bromide Speciation by HPLC-ICP-MS, presentation at PITTCON, Orlando, Florida, March 2006. 

(8)  K.R. Neubauer, W.M. Reuter, and P.A. Perrone, Application Note 007303A_01, PerkinElmer Corporation, Shelton, Connecticut (2006). 

(9)  K.R. Neubauer, W.M. Reuter, P.A. Perrone, and Z.A. Grosser, Application Note 007050_01, PerkinElmer Corporation, Shelton, Connecticut (2004). 

Bottled water has become increasingly popular over the past several years for convenience and safety. In some areas where publicly supplied tap water is contaminated or contains bacteria, this assumption is valid. However, in areas with clean tap water, the presence of bottled water can be controversial because it might be less clean than the local tap. This article discusses the analysis of inorganic contaminants in bottled water, including regulated contaminants and bromate. Detection limit considerations and speed of analysis also are discussed.

Lee Davidowski

Articles

ICP-MS with Autodilution and Autocalibration for Implementing the New USP Chapters on Elemental Impurities

Bottled Water: Popular, But Is It Safe?