SURVEY OF QUALITY REGULATIONS AND STANDARDS

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Using Spectrophotometry for Quality Control

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

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Using Spectrophotometry for Quality Control

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.

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Using Spectrophotometry for Quality Control

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

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Using Spectrophotometry for Quality Control

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?

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Using Spectrophotometry for Quality Control

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 g/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.

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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 C6H5NO2) 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 g/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 A237/A262 ratio for the RS. 6.1.11.4. The A237/A262 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

g/mL.

6.2.1. Weigh out 200 mg of niacin TS. Use a calibrated analytical balance.

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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 g/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 A262 nm and A237 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 (A237/A262) for the TS. 7.1.1.2.1. If the A237/A262 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 (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 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

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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 A237 ______________ RS A262 _________ RS A237/A262 ______________

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 A237 ______________

TS A262 _________TS A237/A262 ______________

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 A237 ______________

TS A262 _________TS A237/A262 ______________

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) ________________

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