Creative Ideas for Teaching GMP
By Madison Area Technical College Biotechnology Department
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USING ABSORPTION SPECTRA FOR
QUALITY CONTROL IN A PHARMACEUTICAL COMPANY
Authors: Noreen Warren, Diana Brandner, and Lisa A. Seidman
Madison Area Technical College
Madison, WI 53704
July 18, 2000
This laboratory exercise introduces students to SOPs and to an example of a quality control assay. NOTE: This exercise requires access to a properly calibrated UV scanning spectrophotometer. Also, this exercise assumes students have some previous familiarity with spectrophotometry.
Before a product can be released for sale it is necessary to test whether it meets its specifications. Spectrophotometry is sometimes used to analyze drug products as one method of testing their identity, purity, and concentration. This exercise is intended to demonstrate the use of spectrophotometry in quality control. In this laboratory exercise you will:
A. GENERAL INFORMATION ABOUT ABSORBANCE SPECTRA
An absorbance spectrum is a plot of how much light a sample absorbs versus the wavelength of that light. To produce such a spectrum, a sample is placed in a spectrophotometer and its absorbance is measured successively at different wavelengths. When such a spectrum is prepared, wavelength is on the X axis and absorbance is on the Y axis. Most modern spectrophotometers can be operated in such a way that they automatically scan the absorbance of the sample at different wavelengths and print out a plot.
Qualitative applications of spectrophotometry use the spectral features of a sample to obtain information about the nature of the sample's component(s). Sometimes it is possible to use an absorbance spectrum like a "fingerprint" to identify an unknown substance. Spectra of organic compounds produced using infrared (IR) light are complex and form particularly distinctive "fingerprints". Organic chemists therefore frequently use IR spectra to identify compounds.
UV/Visible spectra are less complex and distinctive than IR spectra and so are used less commonly for identification of unknown substances. There are, however, situations where information about a sample can be obtained from its UV/Visible spectrum. For example,The U.S. Pharmacopeia (discussed below) includes a number of tests of pharmaceuticals in which an absorbance spectrum from 200 to 400 nm is used to confirm the identity of a drug. In these identity tests the spectrum of the test sample is compared to the spectrum of a pure reference material prepared under identical conditions. If the sample and reference have identical spectra, it is likely that they are the same compound. UV/Vis identity tests are frequently used because they are simple, rapid, and the equipment to prepare an absorbance spectrum is widely available. A problem with identifying substances using UV/Visible spectrophotometry is that some compounds whose structures are similar have spectra that cannot be distinguished. Therefore, UV spectra are frequently used in conjunction with other identification methods.
Niacin is a vitamin, also known as Vitamin B3, that is widely available in pill form from health food and drug stores. Niacin is essential for humans and the recommended daily dose is: 19 mg (males), 14 mg (females), and 20 mg (pregnant or lactating women). Niacin is sometimes prescribed in high dosages to lower cholesterol. People also take niacin supplements because they think it helps ease the severity of migraine headaches and alleviates gastrointestinal disturbances.
When consumers purchase niacin supplements they expect that the pills will indeed contain niacin and will be uncontaminated by unexpected materials. They also expect that the tablets will contain a specific amount of niacin, as shown on the label. Manufacturers need to have methods to assay their product to be sure the niacin is present and uncontaminated. In this exercise, we will use a spectrophotometric method to test niacin tablets purchased at local stores. We will check samples to see that they do indeed contain niacin (this is a test of identity). We will also look at whether contaminants are present (this is a test of purity), and we will test the concentration of niacin in the tablets. Note that the spectrophotometric method we are using would normally be performed in conjunction with other assays. A spectrophotometric method, such as this one, is seldom sufficient to determine with certainty the components of a drug product.
C. THE U.S. PHARMACOPEIA
The particular analytical method we will be using is derived and modified for teaching purposes from theUSP (United States Pharmacopeia). The USP is a compilation of officially recognized standard methods relating to medicines and other health care technologies. This massive (more than 1000 page) book includes an alphabetical listing of drug substances, dosage forms, and pharmaceutical ingredients. For each item, it provides standards, tests, assays, and specifications that are intended to measure the strength, quality, and purity of that product/entity. Every pharmaceutical manufacturer who sells products in the US must use the analytical methods in the USP, or be prepared to justify with extensive evidence why another method is suitable.
Read the entire SOP and ask any questions you might have.
Decide how to document that you have performed this SOP correctly. You may choose to prepare a form on which you will record your data, or you may use your laboratory notebook.
Your instructor will provide you with standards and samples to test using the SOP below.
1.0 Purpose. This SOP describes the quality control procedures to verify the identity, purity, and concentration of niacin in a preparation.
2.0 Scope. This SOP applies to testing of in-process samples and final lots of niacin containing products.
3.0 Responsibility. Technicians in the quality control department.
4.1 USP/NF monograph pages 1080-1081
5.1 Reference Standard (RS): A chemical that is known to have certain properties and which can be used to evaluate the properties of a sample. In this case, the RS is highly purified niacin.
5.2 Test Sample (TS): The material(s) whose properties are to be tested, in this case, niacin from drug and grocery stores.
6.0 Reagents and materials
6.1 Niacin reference standard (Sigma catalog #N5410 CAS No. 59-67-6; niacin is C6H5NO2) (For teaching purposes, the Sigma product is suitable. More expensive reference standards are used in the pharmaceutical industry.)
6.2 UV scanning spectrophotometer, calibrated within past year
6.3 Quartz cuvettes, a matched pair
6.4 Analytical balance
6.5 Ultrapurified water
6.6 500 mL and 100 mL volumetric flasks
7.0 Hazard communication. No special safety precautions required. Wear customary lab coat and safety glasses.
8.1 Prepare the niacin reference standard (RS)
8.1.1 Weigh out 200 mg of niacin RS
18.104.22.168 Use the calibrated analytical balance
8.1.2 Transfer to a 500 mL volumetric flask
8.1.3 Dissolve in ultrapurified water
8.1.4 Mix thoroughly on a stir plate
8.1.5 Remove stir bar
8.1.6 Bring to volume of 500 mL (concentration is now 0.4 mg/mL)
8.1.7 Label as ARS, 0.4 mg/mL@ along with date, preparer=s name, and source
22.214.171.124 This stock solution of RS is supposed to be stable for 6 months
8.1.8 The day of assay, remove exactly 5.0 mL of prepared solution and transfer to a 100 mL volumetric flask
8.1.9 Dilute to volume with ultrapurified water (concentration is now 20 mg/mL)
8.1.10 Label as reference solution (RS, 20 mg/mL)
8.1.11 Check the RS
126.96.36.199 Blank the spectrophotometer using ultrapurified water in a quartz cuvette
188.8.131.52 Determine the absorbance at 237 nm and at 262 nm for the RS
184.108.40.206 Calculate the A237/A262 ratio for the RS
220.127.116.11 The A237/A262 ratio must be between 0.35 and 0.39, otherwise, prepare a new RS and begin again
8.2 Prepare the niacin test sample(s) (TS) that are to be assayed to 20 mg/mL
8.2.1 Calculate the amount of tablet that contains 200 mg of niacin
18.104.22.168 Weigh the tablet on a calibrated analytical balance (TW)
22.214.171.124 Determine the amount of niacin in the tablet based on the bottle label (AN)
126.96.36.199 Divide the weight (TW) by the amount of niacin (AN):
TW/AN = C
188.8.131.52 Determine the weight of sample (T) required to get 200 mg:
(200 mg)(C) = T
8.2.2 Grind the tablet to a fine powder with a mortar and pestle
8.2.3 Weigh out the required amount (T) of the niacin TS
184.108.40.206 Use the calibrated analytical balance
8.2.4 Transfer to a 500 mL volumetric flask
8.2.5 Dissolve in ultrapurified water
8.2.6 Mix thoroughly on a stir plate
8.2.7 Remove stir bar
8.2.8 Bring to volume of 500 mL (concentration is now 0.4 mg/mL)
8.2.9 Remove exactly 5.0 mL of prepared solution and transfer to a 100 mL volumetric flask
8.2.10 Dilute to volume with ultrapurified water (concentration is now 20 mg/mL)
8.2.11 Label as test solution (TS); include your name, date, and source of sample including lot number, catalog number, brand, and other identifying information
8.3 Prepare absorbance spectra for both reference and test samples
8.3.1 Using water in a quartz cuvette as a blank, blank the spectrophotometer from 200 nm to 400 nm
8.3.2 Transfer the RS to a cuvette
220.127.116.11 Clean and use the same cuvette as was used for the blank, or use a previously matched cuvette for the RS.
8.3.3 Prepare an absorbance spectrum for the RS from 200 to 400 nm.
8.3.4 Transfer the TS to a cuvettte
18.104.22.168 Clean and use the same cuvette as was used for the RS, or use a previously matched cuvette for the TS.
8.3.5 Prepare an absorbance spectrum for the TS
8.3.6 Repeat steps 8.3.4-8.3.5 for each TS
8.4 Determine the absorbances at 262 nm and 237 nm for both the reference and test samples
8.4.1 Using ultrapurified water in a quartz cuvette, blank the spectrophotometer at 262 nm
8.4.2 Transfer the RS to a cuvette
22.214.171.124 Clean and use the same cuvette as was used for the blank, or use a previously matched cuvette for the RS.
8.4.3 Determine and record the absorbance of the RS at 262 nm
8.4.4 Transfer the TS to a cuvettte
126.96.36.199 Clean and use the same cuvette as was used for the RS, or use a previously matched cuvette for the TS.
8.4.5 Determine the absorbance of the TS at 262 nm
8.4.6 Repeat steps 8.4.4-8.4.5 for each TS
8.4.7 Using ultrapurified water in a quartz cuvette, blank the spectrophotometer at 237nm
8.4.8 Repeat steps 8.4.2 - 8.4.6 for the RS and each TS at this wavelength
9.0 Analysis of Data
9.1 Qualitative analysis
9.1.1 Compare the spectra for the RS and each TS
188.8.131.52 Determine the absorbance maxima and minima; see attached example
184.108.40.206.1 If the maxima and minima or the RS and TS are at the same wavelength (+ 2 nm) they are within specification, otherwise, reject the TS
220.127.116.11 Determine the ratio of absorbance at 237 and 262 (A237/A262) for the TS
18.104.22.168.1 If the A237/A262 ratio for the TS is between 0.35 and 0.39, accept the sample, otherwise, reject it
9.2 Quantitative analysis
9.2.1 Calculate the concentration of the niacin in the TS using the following equation: Concentration niacin = C (ATS/ARS)
where C = concentration of niacin in the RS
ATS = the absorbance of the niacin TS at 262 nm
ARS = the absorbance of the niacin RS at 262 nm
9.2.2 Compare the value for the TS to the specifications on the label of the product
1. Compare the shapes of the spectra for your test samples to the reference standard. Also compare the maxima and minima for each spectrum. What do you think these spectra tell you about the identity of your test samples?
2. Did the concentration of niacin in the test samples match the concentration specified on their label? Show and explain your calculations. Comment on the purity of the niacin samples.
3. What factors may have affected the accuracy of your test results? Consider, for example, the preparation of the samples and the calibration of the spectrophotometers. What might happen if your used two spectrophotometers; one for the RS and the other for the TS?
This exercise requires a calibrated, scanning UV spectrophotometer for each group of students. It also assumes that students have some familiarity with spectrophotometry. Therefore, we recommend this exercise for college classes. Assuming that the samples and the standard are all prepared in advance, and that different groups of students prepare spectra of different samples, the exercise can be completed in one three hour laboratory period.
To prepare for this laboratory, we suggest having the students create a form on which they will record their data. This will help them focus on the outcomes of the procedure. Make sure the form contains information including: name of person writing form, who approved form (teacher should be OK), the analysts names, date the form was written, date work was done, all information about the RS and TS, the model and some ID for the spectrophotometer used, the absorbances at the relevant wavelengths, the location of spectra (stapled to form should work), etc.
The test samples should be weighed out to give 200 mg, based on the information on the label. For example, if a label states that a tablet contains 250 mg of niacin, and the tablet weighs 500 mg, then there is 1 mg of niacin in every 2 mg of tablet. Grind up the tablets as finely as possible and use 400 mg of the powder. Note that the tablets take a while to dissolve.
We found that the samples are not stable when dilute, however, the 0.4 mg/mL stocks appeared to be stable at least four months. The A237/A262 for our sample, freshly diluted, ranged from 0.36 to 0.38, as expected. The spectra for the fresh sample had a maximum between 260 nm and 262 nm. It has a minimum at about 238 nm; as shown on the attached plot. In contrast, the maxima and minima were different for the diluted samples that had been stored and a new peak appeared, as shown on the attached plot.
MATERIALS AND EQUIPMENT
NSF Award #9850325