LABORATORY EXERCISE: USING SPECTROPHOTOMETRY FOR QUALITY CONTROL: NIACIN

SURVEY OF QUALITY REGULATIONS AND STANDARDS LABORATORY EXERCISE: USING SPECTROPHOTOMETRY FOR QUALITY CONTROL: NIACIN Submitted by Madison Area Technical College Contact Person: Lisa Seidman, Lseidman@matcmadison.edu February, 2013

This activity is excerpted from Laboratory Manual for Biotechnology and Laboratory Science: The Basics, by Seidman, Kraus, Brandner, and Mowery, Published by Benjamin Cummings, 2011. ISBN 0-321-64402-6. It is reproduced here for instructional use by Bio-Link educators.

SCENARIO Working as a Quality Control Analyst You work in the quality control (QC) department in a nutraceutical company that is trying to compete in the lucrative vitamin market. You have been provided with a standard operating procedure (SOP) for an assay to determine the quality of niacin tablets. Your job now is to become familiar with this assay and to use it to test the niacin tablets of several of your competitors. In this laboratory exercise, you will use UV spectrophotometry to perform QC assays on niacin tablets. You will compare the spectra of niacin from several companies with the spectrum of a niacin standard solution to see if they are the same. This comparison is a qualitative assay of identification. You will also calculate the concentration of niacin in the purchased tablets, which is a quantitative assay. In this QC situation, a form is provided for documentation purposes, and you will not use your laboratory notebook. In this exercise you will achieve the following objectives: Practice following an SOP exactly. Learn about the use of spectrophotometry in a pharmaceutical quality control setting. Practice a spectrophotometric assay that is both qualitative and quantitative. Practice using a form for documentation purposes. BACKGROUND A. USP NIACIN ASSAY Niacin is an essential vitamin that is widely available in drug and health food stores. Niacin is sometimes prescribed in high dosages to lower cholesterol. People also take niacin supplements because they think niacin helps ease the severity of migraine headaches and alleviates gastrointestinal disturbances. Consumers who purchase niacin supplements expect that the pills will, indeed, contain niacin in the amount shown on the label. They also expect that the tablets will not be contaminated with unexpected materials. Manufacturers need to have methods to assay their niacin tablets to be sure that they meet these requirements. In this exercise, you will use a spectrophotometric method to test niacin tablets. This assay will allow you to see if the tablets

do indeed contain niacin. This is a qualitative test of identity. This assay will also detect whether certain contaminants are present. This is a qualitative test of purity. The assay will also allow you to test the concentration of niacin in the tablets. Figure 1 shows student results when performing this assay. The particular method you will be using is derived from the United States Pharmacopeia (USP). The USP is a compilation of officially recognized standard methods relating to medicines. The methods in the USP are mandatory in the pharmaceutical industry and other situations where a company is compliant with current Good Manufacturing Practices (cgmp). The USP describes UV assays for drugs in a general way as follows (USP 23, Rockville, MD: United States Pharmacopeial Convention, Inc., 1994, p.1724): Ultraviolet Absorption a test solution and a standard solution are examined spectrophotometrically, in 1 cm cells [cuvettes], over the spectral range from 200 to 400 nm... Dissolve a portion of the substance under examination in the designated Medium to obtain a test solution having the concentration specified...for Solution. Similarly prepare a Standard solution containing the corresponding USP Reference Standard. Record and compare the spectra concomitantly obtained for the test solution and the Standard solution. Calculate absorptivities and/or absorbance ratios where these criteria are included. The requirements are met if the ultraviolet absorption spectra of the test solution and the standard solution exhibit maxima and minima at the same wavelengths and/or absorbance ratios are within specified limits. As you perform this laboratory exercise, note how the tasks you perform match this general description from the USP. B. STABILITY TESTING Have you ever opened your medicine cabinet to get an aspirin or cold medication and noticed that the drug you want is expired? The FDA requires expiration dates on drug products because drugs degrade over time. Pharmaceutical manufacturers must perform stability tests to establish how long a drug remains safe and effective. Stability testing can involve simply waiting various lengths of time and then testing the performance and chemistry of the drug. It is also possible to accelerate the stability-testing process by subjecting the product to heat, light, altered ph, etc. Your instructor may have you experiment with accelerated stability tests on niacin by using this spectrophotometric assay. After ensuring that a niacin solution has the proper spectral features, you can try subjecting it to light and/or heat to see if you can detect changes in the product. If you do detect changes, think about what this means in terms of the proper storage of the product. What happens, for example, to the product if a consumer stores it in a hot kitchen?

a. b. Figure 1. Niacin Standard. a. The higher spectrum is niacin reference standard freshly diluted from 0.4 mg/ml stock. The absorbance peak maximum is close to 262 nm, and the minimum UV absorbance is close to 237 nm. The second niacin preparation was purchased at the store. It has a similar pattern of absorbance to the standard, although the height of the niacin peak is somewhat lower than that of the reference standard. Both niacin preparations pass the test for niacin in the SOP. b. Degraded niacin standard. Diluted niacin reference standard material was stored four months at a concentration of 20 μg/ml in a clear bottle. The absorbance peak maximum is at about 250 nm, and the minimum is at about 221 nm. An extra peak appears. This indicates that niacin degrades with storage. PLANNING YOUR WORK: NIACIN QUALITY CONTROL Plan your work before coming to lab by using a method that you have found to be effective. LABORATORY PROCEDURE Follow this SOP exactly as it is written. If you have questions, ask your supervisor (instructor) before proceeding.

CLEANEST GENES CORPORATION page 1 of 2 SOP #S708, revision 01 SOP For the Qualitative and Quantitative Analysis of Niacin (Nicotinic Acid)* Written by Noreen Warren Date 8/11/10 Approved by Lisa Seidman Date 9/2/10 Approved by Diana Brandner Date 9/6/10 1. Purpose. This SOP describes the quality control procedures to verify the identity, purity, and concentration of niacin. 2. Scope. This SOP applies to testing of in-process samples and final lots of niacin products. 3. Responsibility. Analysts in the quality control department. 4. Reagents and materials. 4.1. Niacin reference standard (Sigma catalogue #N5410 CAS No. 59-67-6; niacin is C 6 H 5 NO 2 ) 4.2. UV spectrophotometer, properly calibrated 4.3. Quartz cuvettes, preferably a matched pair 4.4. Analytical balance, properly calibrated 4.5. Ultrapurified water 4.6 500 ml and 100 ml volumetric flasks 5. Associated Forms. Record results on Form F708. 6. Procedures. 6.1. Prepare the niacin reference standard (RS). 6.1.1. Weigh out 200 mg of niacin RS with a calibrated analytical balance. 6.1.2. Transfer to a 500 ml volumetric flask. 6.1.3. Dissolve in ultrapurified water. 6.1.4. Mix thoroughly on a stir plate. 6.1.5. Remove stir bar. 6.1.6. Bring to volume of 500 ml (concentration is now 0.4 mg/ml). 6.1.7. Label as RS, 0.4 mg/ml along with date, preparer s name, and source. This stock solution of RS should be stable for six months if stored in the dark. 6.1.8. On the day of assay, remove exactly 5.0 ml of prepared solution and transfer to a 100 ml volumetric flask. 6.1.9. Dilute to volume with ultrapurified water (concentration is now 20 μg/ml). 6.1.10. Label as reference standard (RS). 6.1.11. Check the RS. 6.1.11.1. Blank the spectrophotometer using ultrapurified water in a quartz cuvette. 6.1.11.2. Determine the absorbance at 237 nm and at 262 nm for the RS, using the same cuvette as was used for the blank or using a matched cuvette. 6.1.11.3. Calculate the A 237 /A 262 ratio for the RS. 6.1.11.4. The A 237 /A 262 ratio must be between 0.35 and 0.39; otherwise, prepare a new RS and begin again. 6.2. Prepare the niacin test samples (TSs) that are to be assayed to a concentration of 20 μg/ml. 6.2.1. Weigh out 200 mg of niacin TS. Use a calibrated analytical balance.

SOP #S708, revision 01 CLEANEST GENES CORPORATION page 2 of 2 6.2.2. Transfer to a 500 ml volumetric flask. 6.2.3. Dissolve in ultrapurified water. 6.2.4. Mix thoroughly on a stir plate. 6.2.5. Remove stir bar. 6.2.6. Bring to volume of 500 ml (concentration is now 0.4 mg/ml). 6.2.7. Remove exactly 5.0 ml of prepared solution and transfer to a 100 ml volumetric flask. 6.2.8. Dilute to volume with ultrapurified water (concentration is now 20 μg/ml). 6.2.9. Label as a TS; include your name, date, and source of sample. 6.3. Prepare absorbance spectra for both the RS and TSs. 6.3.1. Using water as a blank in a quartz cuvette, blank the spectrophotometer from 200 nm to 400 nm. 6.3.2. Transfer the RS to a cuvette. 6.3.2.1. Clean and use the same cuvette as was used for the blank, or use a previously matched cuvette for the RS. 6.3.3. Prepare an absorbance spectrum for the RS from 200 nm to 400 nm. 6.3.4. Transfer the TS to a cuvette. 6.3.4.1. Clean and use the same cuvette as was used for the RS, or use a previously matched cuvette for the TS. 6.3.5. Prepare an absorbance spectrum for the TS. 6.3.6. Repeat steps 6.3.4-6.3.5 for each TS. 6.4. Determine the A 262 nm and A 237 nm for the reference and test samples. 6.4.1. Using ultrapurified water in a quartz cuvette, blank the spectrophotometer at 262 nm. 6.4.2. Transfer the RS to a cuvette. 6.4.2.1. Clean and use the same cuvette as was used for the blank, or use a previously matched cuvette for the RS. 6.4.3. Determine the absorbance at 262 nm and 237 nm. 6.4.4. Transfer the TS to a cuvette. 6.4.4.1. Clean and use the same cuvette as was used for the RS, or use a previously matched cuvette for the TS. 6.4.5. Determine the absorbance at 262 nm and 237 nm. 6.4.6. Repeat steps 6.4.4-6.4.5 for each TS. 7. Analyze the Data. 7.1. Perform qualitative analysis. 7.1.1. Compare the spectra for the RS and each TS. 7.1.1.1. Determine the absorbance maxima and minima. 7.1.1.1.1. If the maxima and minima of the RS and TS are at the same wavelength (+ 2 nm) they are within specification, otherwise, reject the TS. 7.1.1.2. Determine the ratio of absorbance at 237 nm and 262 nm (A 237 /A 262 ) for the TS. 7.1.1.2.1. If the A 237 /A 262 ratio for the TS is between 0.35 and 0.39, accept the sample; otherwise, reject it. 7.2 Perform quantitative analysis. 7.2.1. Calculate the concentration of the niacin in the TS by using the following equation: Concentration niacin = C (A TS /A RS ) where C = concentration of niacin in the RS A TS = the absorbance of the niacin TS at 262 nm A RS = the absorbance of the niacin RS at 262 nm 7.2.2. Compare the value for the TS to the specifications on the product label. *Derived from: USP/NF 2000, monograph pages 1080-1081 and 1724

CLEANEST GENES CORPORATION Form #F708, revision 01 page 1 of 1 FORM FOR SOP FOR THE QUALITATIVE AND QUANTITATIVE ANALYSIS OF NIACIN (NICOTINIC ACID) Written by Noreen Warren Date 8/11/10 Approved by Lisa Seidman Date 9/2/10 Approved by Diana Brandner Date 9/6/10 FORM #F708 Scope: This form is to be used to record information with SOP # S708, revision 01: SOP for the Qualitative and Quantitative Analysis of Niacin (Nicotinic Acid) Date Name of analyst Signature of analyst Reference Standard (RS) ID number Date prepared Date diluted RS A 237 RS A 262 RS A 237 /A 262 RS Pass or Fail (Is it within 0.35-0.39?) (Do not continue if RS does not pass) RS Absorbance Maximum nm RS Absorbance Minimum nm Test Sample (TS) ID number Brand Name Date prepared Date diluted TS A 237 TS A 262 TS A 237 /A 262 TS Pass or Fail (Is it within 0.35-0.39?) TS Absorbance Maximum Pass/Fail (within + 2 nm of RS?) TS Absorbance Minimum Pass/Fail (within + 2 nm of RS?) TS concentration (show calculation) Test Sample (TS) ID number Brand Name Date prepared Date diluted TS A 237 TS A 262 TS A 237 /A 262 TS Pass or Fail (Is it within 0.35-0.39?) TS Absorbance Maximum Pass/Fail (within + 2 nm of RS?) TS Absorbance Minimum Pass/Fail (within + 2 nm of RS?) TS concentration (show calculation)

LAB MEETING/DISCUSSION QUESTIONS 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? 4. Report on your results if you were able to perform a stability study. 5. What is the purpose of quality control assays? How can spectrophotometry be used in a quality control setting for both qualitative and quantitative purposes?

NOTES TO THE INSTUCTOR: This exercise models a pharmaceutical quality control assay. This assay does not rely on a standard curve but rather is based on the absorbance of a single standard. It is critical that this standard be accurately weighed out and be totally dissolved. The spectrophotometer used for this assay must be calibrated; otherwise the peaks may be shifted and the desired A 237 /A 262 ratios cannot be achieved for standards or samples. This exercise is interesting to use simply to test different brands of niacin. As you can see in this table, one of the brands we analyzed did not appear to contain any niacin at all. This may be due to a problem with the product, an error on our part, or overly-long or improper storage. Student Intern Data for Three Types of Niacin Sample ID A 237 A 262 A 237 /A 262 Pass/Fail RS Niacin 0.2472 0.6919 0.3573 Pass TS Twin Lab 0.1352 0.3716 0.3638 Pass TS Country Life -0.0009 0.0015 NA NA This laboratory exercise can also be the basis for student experiments. For example, students might perform stability studies to explore the factors that degrade a niacin product, such as exposure to light, heat, freezing and thawing, and time. The procedure as it is written in the official methods of the United States Pharmacopeia is shown above. Students may be directed to compare this official short-hand procedure with the version in the laboratory manual that is written out as a standard operating procedure (SOP). Students might discuss who writes out such SOPs, how analysts verify that a procedure taken from a publication works in their own laboratory, and the types of details that must be included in an SOP. LAB MEETING/DISCUSSION QUESTIONS 3. What factors may have affected the accuracy of your test results? Dissolving the niacin tables is a critical step and it is important to be sure they are thoroughly dissolved. As noted above, it is essential to have an accurately calibrated spectrophotometer. 5. What is the purpose of quality control assays? How can spectrophotometry be used in a quality control setting for both qualitative and quantitative purposes? This assay provides both qualitative and quantitative information. Students generally can appreciate how an assay like this is used as a relatively rapid test of product quality.