QUALITY CONTROL

Quality-Control Analytical Methods: High-Performance Liquid

Tom Kupiec, PhD of liquid chromatography used to separate and quantify com- Analytical Research pounds that have been dissolved in . HPLC is used to Oklahoma City, Oklahoma determine the amount of a specific compound in a solution. For example, HPLC can be used to determine the amount of in a compounded solution. In HPLC and liquid Introduction chromatography, where the sample solution is in contact with a Chromatography is an based on the sep- second solid or liquid phase, the different solutes in the sample aration of due to differences in their structure solution will interact with the stationary phase as described. and/or composition. In general, chromatography involves The differences in interaction with the column can help sepa- moving a sample through the system over a stationary phase. rate different sample components from each other. The molecules in the sample will have different affinities and interactions with the stationary support, leading to separation Practical Aspects of HPLC Theory of molecules. Sample components that display stronger inter- In order to understand HPLC and to utilize its practical ap- actions with the stationary phase will move more slowly plications effectively, some basic concepts of chromatographic through the column than components with weaker interac- theory are presented here. tions. Different compounds can be separated from each other Chromatographic Principles as they move through the column. Chromatographic separa- Retention. The retention of a drug with a given packing mate- tions can be carried out using a variety of stationary phases, in- rial and eluent can be expressed as a retention time or reten- cluding immobilized silica on glass plates (thin-layer chro- tion volume. Retention or volume is the quantity of the matography), volatile gases (), paper (pa- mobile phase required to pull the sample through the column. per chromatography) and liquids (liquid chromatography). Retention time is defined as how long a component is retained High-performance liquid chromatography (HPLC) is a type in the column by the stationary phase relative to the time it re- sides in the mobile phase. The retention is best described as a Continuing Education column capacity ratio (k´), which can be used to evaluate the GOALS AND OBJECTIVES efficiency of columns. The longer a component is retained by the column, the greater is the capacity factor. The column ca- “Quality-Control Analytical Methods: High-Performance pacity ratio of a compound (A) is defined by the following Liquid Chromatography” equation: Goal: The goal of this presentation is to provide compounding T −T V −V pharmacists with auxiliary information concerning the use of high- k´ = A O = A O performance liquid chromatography for quality control of com- TO VO pounded preparations. where VA is the elution volume of component A and V0 is the Objectives: After reading and studying the article, the reader will elution volume of a nonretained compound. At constant flow be able to: rate, retention times (TA and T0) can be used instead of reten- 1. Discuss basic theory behind chromatographic separation. tion or elution volumes. 2. Discuss basic instrumentation and components of high- Resolution. Resolution is the ability of the column to separate performance liquid chromatography. peaks on the chromatograph. Resolution (R) is expressed as the 3. Discuss different analysis methods for high-performance liquid ratio of the distance between two peak maxima to the mean chromatography. value of the peak width at the base line 4. Discuss validation and maintenance of high-performance liquid (T −T )2 R= B A chromatography. wA +wB To complete this continuing education program, go to www.ijpc.com. The where TB is the retention time of component B, TA is the re- program is presented by the IJPC in partnership with P*ceutics Institute @ tention time of component A, wA is the peak width of compo- PCCA, which is accredited by the American Council on Pharmaceutical nent A and wB is the peak width of component B. If R is equal Education as a provider of continuing pharmaceutical education. Upon to or more than 1, then components are completely separated, successful completion of 70 percent of the questions and completion of the but if R is less than 1, then components overlap. evaluation, an ACPE statement of credit for one (0.1 CEU) credit Sensitivity. Sensitivity is a measure of the smallest detectable will be immediately available for printing. The cost is $15. level of a component in a chromatographic separation and is dependent on the signal-to-noise ratio in a given detector.

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the eluent and is coated on an inert support. exchange Figure 1. Schematic of a High-Performance Liquid chromatography has a stationary phase with an ionically Chromatograph. charged surface that is different from the charge of the sample. The technique is based on the ionization of the sample. The stronger the charge of the sample, the stronger the attraction to the stationary phase; therefore, it will take longer to elute off the column. Size exclusion is as simple as screening sam- Mobile Pump Injector Column Detector Data ples by molecular size. The stationary phase consists of mate- Phase for Display rial with precisely controlled pore size. Smaller particles get Reservoir Sample caught up in the column material and will elute later than larg- er particles. Several other types of chromatographic separation have been described, including ion-pair chromatography, Sensitivity can be increased by of the compound which is used as an alternative to ion-exchange chromatogra- of interest, optimization of chromatographic system or minia- phy and chiral chromatography (to separate enantiomers). turization of the system. Instrumentation Chromatographic Mechanisms As shown in the schematic diagram in Figure 1, HPLC in- Systems used in chromatography are often categorized into strumentation includes a pump, injector, column, detector and one of four types based on the mechanism of action, adsorp- integrator or acquisition and display system. The heart of the tion, partition, ion-exchange and size exclusion. system is the column where separation occurs. Since the chromatography arises from interactions between solutes and stationary phase may be composed of micron-sized porous par- the surface of the solid stationary phase. Partition chromatog- ticles, a high-pressure pump is required to move the mobile raphy involves a liquid stationary phase that is immiscible with phase through the column. The chromatographic process be- gins by injecting the solute into the injector at the end of the column. Separation of components occurs as the analytes and mobile phase are pumped through the column. Eventually, each component elutes from the column as a peak on the data For Sterile Compounding display. Detection of the eluting components is important, and Why Build a cleanroom? the method used for detection is dependent upon the detector used. The response of the detector to each component is dis- played on a chart recorder or computer screen and is known as a chromatogram. To collect, store and analyze the chromato- graphic data, integrators and other data-processing equipment are frequently used. Mobile Phase and Reservoir The type and composition of the mobile phase affects the separation of the components. Different are used for different types of HPLC. For normal-phase HPLC, the sol- MicroSphereTM 4x2 vent is usually nonpolar, and, in reverse-phase HPLC, the sol-

MicroSphereTM 8x3 vent is normally a mixture of water and a polar organic sol- TM vent. The purity of solvents and inorganic salts used to make Now, MicroSphere the mobile phase is paramount. A general rule of thumb is to The cleaner, safer, more cost-effective AMERICA’S # 1 use the highest purity of that is available and practical microenvironment for sterile compounding BARRIER ISOLATOR depending on the particular application. The most common and manipulating cytotoxic agents. solvent reservoirs are as simple as glass with tubing connecting them to the pump inlet. CONSIDER THE ADVANTAGES Pumps NO CLEANROOM TO BUILD, NO GOWNING -Very cost-efficient High-pressure pumps are needed to push the mobile phase ASEPTIC - Class 10 - meets all regulations SAFE - Physical barrier between products and operators through the packed stationary phase. A steady pump pressure ERGONOMIC - Easy-to-operate, install and maintain (usually about 1000–2000 psi) is needed to ensure repro- MODULAR - Can be custom-designed- Many sizes/ options available ducibility and accuracy. Pumps are typically known to be ro- bust, but adequate maintenance must be performed to main- tain that characteristic. Inability to build pressure, high TEL: 1-800-ISO-2010 FAX: (514) 956-1032 pressures or leakage could indicate that the pump is not func- E-MAIL: [email protected] tioning correctly. Proper maintenance of the pump system will WEB SITE: isotechdesign.com minimize down time. INNOVATORS IN CLEAN AIR

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Injectors materials with C1, C2, C4, C6, C8 and C22 coatings are also The injector can be a single injection or an automated injec- available. Miscellaneous chemical moieties bound to silica, as tion system. An injector for an HPLC system should provide well as polymeric packing, are designed for purification of spe- injection of the liquid sample within the range of 0.1-100 mL cific compounds. Other types of column packing materials in- of volume with high reproducibility and under high pressure clude zirconia, polymer-based and monolithic columns. (up to 4000 psi). For liquid chromatography, liquid samples Theoretical plates relate chromatographic separation to the can be directly injected and solid samples need only to be di- theory of and are a measure of column efficiency. luted in the appropriate solvent. The number of theoretical plates (n) can be determined by the following equation Detectors 2 tR There are many different types of detectors that can be used n = 16 1 w for HPLC. The detector is used to sense the presence of a ( ) where t is the total retention time and w is the band width of compound passing through and to provide an electronic signal R1 the peak. to a data-acquisition device. The main types of detectors used In general, LC columns are fairly durable with a long service in HPLC are refractive index (RI), (UV-Vis) and life unless they are used in some manner that is intrinsically fluorescence, but there are also diode array, electrochemical destructive—for example, with highly acidic or basic eluents or and conductivity detectors. Each detector has its assets, with continual injections of “dirty” biological or crude sam- limitations and sample types for which it is most effective. ples. Column degradation is inevitable, but column life can be Most applications in drug analysis use detectors that respond prolonged with proper maintenance. Flushing a column with to the absorption of ultraviolet radiation (or visible light) by mobile phase of high elution strength following sample runs is the solute as it passes through the flow- inside the detector. essential. When a column is not in use, it is capped to prevent The recent development of the so-called hyphenated tech- it from out. Particulate samples need to be filtered and niques has improved the ability to separate and identify multi- when possible a guard column should be utilized. Column re- ple entities within a mixture. These techniques include liquid generation could instill some life into a column, but preventive chromatography- (LC-MS), liquid chro- maintenance is the key to preventing premature degradation. matography-mass spectrometry-mass spectrometry (LC-MS- MS), liquid chromatography-infrared (LC-IR) Separation Techniques and liquid chromatography-nuclear magnetic resonance (LC- NMR). These techniques usually involve chromatographic Isocratic versus Gradient Elution separation followed by peak identification with a traditional Elution techniques are methods of pumping mobile phase detector such as UV, combined with further identification of through a column. In the isocratic method, the composition of the compound with the MS, IR or NMR. the mobile phase remains constant, whereas in the gradient Data Acquisition/Display Systems method the composition changes during the . The isocratic method is the simplest technique and Since the detector signal is electronic, use of modern data- should be the first choice when developing a separation. Elu- acquisition techniques can aid in the signal analysis. The data- ent gradients are usually generated by combining the pressur- acquisition system of most HPLC systems is a computer. The ized flows from two pumps and changing their individual flow computer integrates the response of the detector to each com- rates with an electronic controller or data system while main- ponent and places it into a chromatograph that is easy to read taining the overall flow rate constant. and interpret. Other more advanced features can also be ap- plied to a chromatographic system. These features include Derivatization computer-controlled automatic injectors, multi-pump gradient Derivatization of samples involves a that controllers and sample fraction collectors. alters the molecular structure of the analyte of interest to im- Columns prove detection. In HPLC, derivatization of a drug is usually unnecessary to achieve satisfactory chromatography. Derivati- The column or stationary phase is the core of any chromato- zation is used to enhance the sensitivity and selectivity of de- graphic system. Columns are commercially available in differ- tection when available detectors are not satisfactory for the ent lengths, bore sizes and packing materials. The use of the underivatized compounds. correct combination of length and packing material in correla- tion with the appropriate mobile phase can assist in the most Quantitative Analysis effective separation of a sample compound. A variety of The quantification methods incorporated in HPLC are bor- column dimensions are available including preparative, nor- rowed mostly from gas chromatography methods. The basic mal-bore, micro- and mini-bore and capillary columns. Differ- theory for quantitation involves the measurement of peak ent column dimensions can be used for different types of sepa- height or peak area. To determine the (conc.) rations and can utilize different packing materials and flow of a compound, the peak area or height is plotted versus the rates. The most widely used packing materials for HPLC concentration of the substance. For peaks that are well re- separations are silica-based. The most popular material is oc- solved, both peak height and area are proportional to the con- tadecyl-silica (ODS-silica), which contains C coating, but 18 centration. Three different calibration methods, each with its

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own benefits and limitations, can be utilized in quantitative this method, an equal amount of an , a com- analysis: external standard (std.), internal standard and the ponent that is not present in the sample, is added to both the method. sample and standard . The internal standard selected External Standard should be chemically similar to, have similar retention time The external standard method (see Figures 2 and 3) is the and derivatize similarly to the analyte. Additionally, it is im- simplest of the three methods. The accuracy of this method is portant to ensure that the internal standard is stable and does dependent on the reproducibility of the injection volume. To not interfere with any of the sample components. The internal perform this method, standard solutions of known concentra- standard should be added before any preparation of the sample tions of the compound of interest are prepared with one stan- so that extraction efficiency can be evaluated. Quantification is dard that is similar in concentration to the unknown. A fixed achieved by using ratios of peak height or area of the compo- amount of sample is injected. Peak height or area is then nent to the internal standard. AreaInternalStd.in known Areaunknown × plotted versus the concentration for each compound. The plot Conc.unknown = () × ( ) (Conc.known) should be linear and go through the origin. The concentration AreaInternalStd.in unknown Areaknown of the unknown is then determined according to the following Validation formula. It is important to utilize a validated LC method when per- Areaunknown Conc.unknown = conc.known forming analysis. Typical analytical characteristics evaluated ()Areaknown in an LC validation include but not are not limited to preci- Internal Standard sion, accuracy, specificity, limit of detection, limit of quantita- Although each method is effective, the internal standard tion, linearity and range. It is important to consider the US method tends to the most accurate and precise results. In Food and Drug Administration (FDA) and United States

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Pharmacopeia-National Formulary (USP- NF) guidelines when validating LC meth- Figure 2. Chromatogram of Reference Standard. ods used for pharmaceutical samples. USP 27-NF 22, section <1225> provides guid- 7.152 – Triamcinolone acetonide Area: 4101.47 ance on validation of compendial meth- 250 ods, including definitions and determina- 200 tion. The International Conference on Harmonisation of Technical Require- 150 ments for Registration of Pharmaceuticals 100 for Human Use (ICH) guidelines provide 2.066 50 suggestions concerning validation of Milliabsorbance Units 1.237 1.916 2.420 pharmaceuticals. Valuable sources of in- 0 formation providing regulatory guidance 123456789 may be found on the FDA website at Time http://www.fda.gov/cder/guidance. Note: This was a 200 ppm (mcg/mL) solution, so the amount (200 ppm) is divided by the area System suitability tests provide an (4101.47 mAU), resulting in amt/area ratio (4.8763e-2) used for sample determination. evaluation for the function of the overall mAU = milliabsorbance units LC system. This includes all components mcg = microgram that make up a system, such as the instru- mL = milliliters ment, reagents, column packing material, ppm = parts per million details of the procedure and even the ana- lyst. These tests imply that all of the com- ponents of a system constitute a single Figure 3. Chromatogram of Sample. system in which the overall function can be tested. System suitability tests are valu- 7.115 – Triamcinolone acetonide Area: 3854.51 able and have been accepted in general 250 application because reliable and repro- 200 ducible chromatographic results are based 150 on a wide range of specific parameters. In most laboratories there is a standard 100 operating procedure that outlines the 50 1.545 2.696 1.413 2.398 3.351 specifications for running a system’s suit- Milliabsorbance Units 0 ability test. For the test, at least five replicate injections are made of a single 123456789 solution that contains 100% of the ex- Time pected active and excipient ingredients Note: This was a 200 ppm (mcg/mL) solution, so the amount (200 ppm) is divided by the area level. The peak response is measured and (4101.47 mAU), resulting in amt/area ratio (4.8763e-2) used for sample determination. the standard deviation of that response mAU = milliabsorbance units should not exceed the limit set by the mcg = microgram testing monograph or 2%, whichever of mL = milliliters the two is the lowest. Using the USP ppm = parts per million method, the tailing factors of the analytes should be determined. The values should Resources not exceed 2. Peak-to-peak resolutions are also determined by 1. Knox JH, Done JN, Fell AF et al. High-Performance Liquid Chromatogra- using the USP calculations, and the value should not be lower phy. Edinburgh: Edinburgh University Press; 1978. than 1.5. The system test is used to ensure the quality of the 2. Simpson CF. Practical High-Performance Liquid Chromatography. Lon- don: Heyden and Son; 1976. data and of the analysis. 3. Pungor E. A Practical Guide to Instrumental Analysis. Boca Raton: CRC Press; 1995. Summary 4. Moffat AC, Osselton MD, Widdop B. Clarke’s Analysis of Drugs and HPLC is currently the most widely used method of quantita- Poisons. London: Pharmaceutical Press; 2004. tive analysis in the pharmaceutical industry and in pharmaceu- tical analysis laboratories. Address correspondence to: Tom Kupiec, PhD, Analytical Research Laboratories, 840 Research Parkway, Suite 546, Oklahoma City, OK 73104. E-mail: [email protected] ■

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