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QUALITY CONTROL

Quality-Control Analytical Methods: Gas

Tom Kupiec, PhD Since GC is a gas-based separation technique, it is limited to Analytical Research components that have sufficient volatility and thermal stability. Oklahoma City, Oklahoma Practical Aspects of Gas Chromatography Theory Introduction To understand GC and effectively use its practical applica- Chromatography is an based on the tions, a grasp of some basic concepts of general chromato- separation of due to differences in their structure graphic theory is necessary. Chromatographic principles, and/or composition. Chromatography involves moving a sam- including retention, resolution, sensitivity and other factors, ple through the system to be separated into its various compo- are important for all types of chromatographic separation, and nents over a stationary phase. The molecules in the sample will were discussed in volume 8, issue 3 (May/June 2004) of the have different interactions with the stationary support, leading International Journal of Pharmaceutical Compounding.1 to separation of similar molecules. Chromatographic separa- The GC section of United States Pharmacopeia (USP) 27 tions can be divided into several categories based on the Chapter <621> outlines the basic theory and separation tech- mobile and stationary phases used, including thin-layer chro- nique of GC. A compound is vaporized, introduced into the matography, gas chromatography (GC), carrier gas and then carried onto the column. The sample is and high-performance liquid chromatography (HPLC). then partitioned between the gas and the stationary phase. The GC is a physical separation technique in which components compounds in a sample are slowed down to varying degrees of a mixture are separated using a mobile phase of inert carrier due to the sorption and desorption on the stationary phase. gas and a solid or liquid stationary phase contained in a col- The of the compound is characterized by the partition umn. The separation is based on the interactions of the vapor- ratio kD′, which is a dimensionless quantity also called the ized components in a mixture with the stationary phase as they capacity factor. The partition ratio can also be thought of as the are moved along by the mobile phase. ratio of the time required for the compound to flow through the column (the retention time) to the elution time of an unre- tained compound. The value of the capacity factor is depen- Continuing Education dent on several elements of the chromatographic system, GOALS AND OBJECTIVES including the chemical nature of the compound; the nature, “Quality-Control Analytical Methods: Gas Chromatography” amount and surface area of the stationary phase; the column Goal: The goal of this presentation is to provide compounding temperature; and the gas flow rate. Capacity factor is essential pharmacists with auxiliary information concerning the use of gas for separation by GC because separation is only possible if the chromatography for quality control of compounded preparations. compounds in the sample have different capacity factors. Objectives: After reading and studying the article, the reader will be able to: Samples and 1. Review the basic theory behind chromatographic separation. A variety of sample types can be successfully analyzed by GC. 2. Discuss the basic instrumentation and components of gas Unlike HPLC, which is used to separate larger molecules, GC chromatography. is best suited for analysis of samples with smaller molecules. 3. Discuss the different analysis methods by gas Another important characteristic of samples for GC analysis is chromatography. that they must be volatile. If a compound is not volatile a tech- 4. Discuss the applications, strengths and limitations of gas nique called can be used to increase its solubility chromatography. To complete this continuing education program, go to www.ijpc.com. The and add to its volatility. Derivatization of samples involves a program is presented by the IJPC in partnership with P*ceutics Institute @ that alters the molecular structure of the an- PCCA, which is accredited by the American Council on Pharmaceutical alyte of interest to improve detection. Different types of com- Education as a provider of continuing pharmaceutical education. Upon pounds called derivatizing agents can be used to increase the successful completion of 70 percent of the questions and completion volatility of sample components. However, if possible it is best of the evaluation, an ACPE statement of credit for one (0.1 CEU) to avoid derivatization to keep the separation simpler. credit will be immediately available for printing. The cost is $15.

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auto sampler system. A major source of precision errors in GC Figure 1: Schematic of a Gas Chromatograph. is poor injection technique. Auto samplers are very effective and help ensure that precisely the same sample volume is in- Heater Oven Heater jected every time, thereby eliminating injection errors. The injector temperature is also important for separation. The temperature of the injector is used to rapidly vaporize the liq- uid sample into a gaseous phase that can be carried to the column for separation. The temperature of the injector site Compressed Injector Column or Detector Recorder can be varied to help optimize separation. Different sample Gas (Carrier Stationary or Computer components will dictate what temperature is necessary Gas) Phase for vaporization. Hardware Carrier Gas In the early days of GC experiments, the carrier gas was seen The hardware components used in typical GC systems in- merely as the mass transport system. However, it is becoming clude an injector, a carrier gas, a column (stationary phase), an clearer that carrier gas is integral to the chromatographic oven, a detector and a recorder or information processor process. Several inert gases can be used as the carrier gas or (Figure 1). Several components have variable settings that can mobile phase of GC. , and are all be used to optimize the analysis of different sample types. common carrier gases. Each carrier gas has its benefits and Injectors systems for which it is best suited. For example, helium is the Introduction of the sample into a GC system is a critical step most common gas used with GC/ systems. in separation. The reproducibility of the amount of sample in- Before the carrier gas can be used, it is important to ensure jected is important to ensure the reproducibility of results. A that it contains no oxygen because oxygen can have detrimen- sample can be injected manually into the system or by using an tal effects on the stationary phase of GC. Also, the chemical nature of the carrier gas has an effect on the efficiency of the GC column. The pressure at which the carrier gas is moving influences the retention time of samples on the column. In- creasing the pressure decreases the retention time. Varying both the carrier gas and the pressure at which the gas is exert- ed on the column can ensure that the sample has ample time to interact with the stationary phase and improve the separation. Column/Stationary Phase The column and stationary phase are responsible for the ma- jority of the separation of sample components. Interaction between the mobile phase, stationary phase and sample com- ponents determines how components are separated, so selec- tion of columns and stationary packing material is critical. There is a great deal of variation in commercially available columns and packing material. Depending on the components to be separated, the mobile phase being used and the desired degree of separation, different combinations of column type, column length and packing material can be used to achieve optimal results. Columns can be classified by column diameter and packing material. The three main types of GC columns are (1) conven- tional, (2) preparative and (3) capillary. Columns can be either packed or open. Packed columns can contain either a porous or nonporous stationary phase. A multitude of different mate- rials are used to pack columns. Each material has its own prop- erties, limitations and effective separation parameters. The capillary column is the most frequently used column for GC separations. Both conventional and capillary columns have ad- vantages and disadvantages. For example, more packing mate- rials are available for conventional columns, but capillary columns give improved sensitivity.

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When selecting a column, a major choice is among nonpolar, that are well resolved, both peak height and area are propor- moderately polar or polar columns. Special-phase columns tional to the concentration. Three different calibration meth- such as chiral columns can also be utilized to separate isomers. ods, each with its own benefits and limitations, can be used in Other important factors to consider are packing material, col- quantitative analysis: (1) the external standard (std), (2) the in- umn length and column diameter. ternal standard (IS) and (3) the method. For Columns can be the most expensive component of a chro- GC, the most commonly used quantitative methods are the IS matographic system, so proper maintenance and use can help and standard addition methods. For our present purpose, only control cost. For proper column care it is best to consult the the IS method is discussed. manufacturer’s guidelines. The IS is an effective method because it tends to the Oven most accurate and precise results of all the quantitative meth- ods. With this method, an equal amount of an IS, a component The column resides in an oven, and temperature, which that is not present in the sample, is added to both the sample greatly affects the effectiveness of the chromatographic separa- and calibrator solutions. The IS selected should be chemically tion, is an extremely important factor used in controlling GC. similar to the analyte and have a similar retention time and In many cases, isothermal (constant temperature) is not the similar derivatization. Additionally, it is important to ensure most effective temperature mode for sample separation; in that the IS is stable and does not interfere with any of the such cases, a temperature program can be used. Most GC sample components. The IS should be added before any prepa- temperature programs have an initial temperature, a ramp ration of the sample so that extraction efficiency can be evalu- (degree increase per minute) and a final temperature. ated. Quantitation is achieved by using ratios of peak height or Detector area of the component to the , as shown in The detector is used to sense the presence of a compound the following formula: passing through and to provide an electronic signal to an inte- AreaIScalibrator × Areasample × grator. A variety of detectors are commercially available to be Conc.sample = () ( ) (Conc.calibrator) AreaIS Areacalibrator used with GC, each having its own limitations and advantages: sample ■ Electron capture (ECD). The ECD is used with organic compounds and has many environmental applications. ■ Flame ionization (FID). The most commonly used detector in GC, FID is typically used with organic compounds and is GROWTH HORMONE widely used in quality-control analysis of pharmaceutical compounds. ■ Mass spectrometry (MSD). The MSD can be coupled with GC as a powerful qualitative component for the identifica- Saizen® tion of compounds. 5mg & 8.8mg ■ Nitrogen phosphorous (NID). This detector is used most commonly for drug analysis in tissues and bodily fluids. ■ Thermal conductivity (TCD). This detector is considered a universal detector and is nondestructive to analytes. Recorder Just as it is with HPLC, the recorder in a GC system serves to convert the information collected by the detector into a for- mat that is understandable. Since the detector signal is elec- ® tronic, the use of modern data acquisition can aid in signal Norditropin Cartridges analysis. The most common data-acquisition technique is 5mg & 15mg cartridges through use of a computer, which integrates the response of the detector to each component and places it into a chromato- FREE Nordipen Kit with purchase of Norditropin graph that is easy to read and interpret. Other more advanced features can also be applied to a chromatographic system. CALL FOR PRICE QUOTES These include computer-controlled automatic injectors, oven temperature programs and carrier gas pressure. 866-HGH-WHSL (866-444-9475) Free delivery on all orders in the continental US. Quantitative Analysis The basic theory for quantitation of sample components in- volves the measurement of peak height or peak area. For peaks Medical Center Pharmaceuticals 1875 Third Avenue, San Diego, CA 92101

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Figure 2 is an example chromatogram produced in the GC Figure 2: Example Chromatogram Illustrating Gas Chromatog- analysis of menthol, with decanol employed as an IS. raphy Analysis of Menthol with Decanol Used as an Internal Alternatively, a is established by plotting Standard. the response ratios of calibrator/IS versus concentration of the calibrators. Sample concentration is then extrapolated from 5.692 – Menthol the regression equation of the calibration curve based on the 7.277 – Decanol Area: 9015.24 350 response ratio of sample/IS.

300 Area: 7447.91 Validation It is important to use a validated GC method when perform- 250 ing analysis. Typical analytical characteristics evaluated in a GC method are similar to those evaluated in a liquid chro- Area Counts 200 matographic validation and may include precision, accuracy, specificity, limit of detection, limit of quantitation, linearity 150 and range, among other factors. It is important to consider the US Food and Drug Administration (FDA) and USP guidelines 02 4 6 8 10 when validating GC methods used for pharmaceutical samples. Time (Minutes) USP 24 Chapter <1225> provides guidance on validation of Note: The area ratio of menthol/decanol was used in determining the compendial methods, including definitions and determination. concentration of the sample. The sample was a 110 ppm (µg/mL) The International Conference on Harmonisation of Technical solution. Requirements for Registration of Pharmaceuticals for Human mL = milliliter ppm = parts per million µg = micrograms Use (ICH) guidelines provide suggestions concerning valida- tion of pharmaceuticals. Valuable sources of information

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providing regulatory guidance may be found on the FDA web- Summary site at http://www.fda.gov/cder/guidance. Both GC and HPLC are widely used methods for the quality System suitability tests provide an evaluation for the function control of both commercially manufactured and compounded of the overall GC system. This includes all pieces that make up pharmaceuticals. a system, such as the instrument, reagents, packing material, details of the procedure and even the analyst. These tests im- Resources ply that all the components of a system constitute a single sys- 1. Jennings W, Mittlefehldt E, Stremple P. Analytical Gas Chromatography. tem for which the overall function can be tested. These tests San Diego, CA: Academic Press. San Diego; 1987. are very valuable and have been accepted in general application 2. McMaster M, McMaster C. GC/MS: A Practical Users Guide. New York, since reliable and reproducible chromatographic results are NY: John Wiley & Sons; 1998. based on a wide range of specific parameters. 3. Keulemans AI. Gas Chromatography. New York, NY: Reinhold Publishing In most laboratories a standard operating procedure outlines Corp.; 1959. 4. Eiceman GA, Hill HH Jr, Gardea-T J. Gas chromatography. Analytical the specifications of running a system suitability test. To evalu- . 2000; 72(12). ate system suitability, at least five replicate injections should be 5. US Pharmacopeial Convention, Inc. United States Pharmacopeia (USP) made of a single solution that contains 100% of the expected 27. Rockville, MD: US Pharmacopeial Convention, Inc.; 2001. active and excipient ingredients levels. The peak response is measured, and the coefficient of variation for five replicates References should not exceed the limit set by the testing monograph, or 1. Kupiec T. Quality-control analytical methods: High-performance liquid 2%. Using the USP method, the tailing factors of the analytes chromatography. IJPC 2004; 8(3): 223-227. should not be greater than 2. Peak-to-peak resolutions (R) are also determined by using the USP calculations and should be Address correspondence to: Tom Kupiec, PhD, Analytical at least 1.5. The system test should be used to ensure the Research Laboratories, 840 Research Parkway, Suite 546, quality of the data and of the analysis. Oklahoma City, OK 73104. E-mail: [email protected] ■

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