Version: This manual was drafted 11/4/2014 · Web viewFor ampicillin, amoxicillin and...

Distributed Pharmaceutical Analysis Laboratory

Draft of procedures for method validation

4 November, 2014

M. Lieberman

1. VERSION: THIS MANUAL WAS DRAFTED 11/4/2014 2

2. CAVEATS 2

3. LIMITATIONS OF THE ANALYTICAL METHODOLOGY 3

4. SAMPLE PREPARATION 4

SAMPLE STORAGE AND TRACKING: 4EXTERNAL STANDARDS: 4PHARMACEUTICAL DOSAGE FORMS: 5

5. METHOD VALIDATION 7

6. SUGGESTED INSTRUMENT PARAMETERS, SOLVENTS, AND GRADIENTS 8

A) AMOXICILLIN 8B) AMPICILLIN 10C) AMOXICILLIN-CLAVULANATE (OR AMOXICILLIN-CLAVULANIC ACID) 11D) CIPROFLOXACIN 13

7. ANALYTICAL METRICS (HOW TO TELL IF YOUR HPLC IS BEHAVING PROPERLY): 14

8. ASSAY AND QUALITY CONTROL 15

1. Version: This manual was drafted 11/4/2014

2. Caveats

Participants in the DPAL must be aware of some legal issues related to pharmaceutical analysis. We report poor quality drugs to the medical regulatory authority (MRA) in the country where the drugs originated, and also to the WHO Rapid Alert system. Since we are doing single tablet analysis, analytical results must be replicated on several samples before triggering a report to the MRA or WHO. If your laboratory turns up a poor quality product that meets these criteria, Prof. Lieberman will work with you to report to the appropriate authorities. Please preserve such samples carefully; in some cases, we may want to subject the sample to LC-MS, or the country MRAs may want to analyze the samples themselves (eg to support legal action).

Second, due to the prevalence of counterfeit and improperly labeled products in developing world markets, it is possible that information such as manufacturer name, lot numbers, or expiration dates on the packaging materials are falsified or missing. Legal counsel at UND recommends that we all use the term "stated to be manufactured by thus and such company" in communications about specific pharmaceutical products. This information must be conveyed to students who work on the analyses.

Third, students must obtain written permission from their instructor before posting pictures of products, analytical data, or other product-specific information in a public forum such as a poster session, news article, web site, or social media posting. Please check that the students adhere to the necessary wording describing the origin of the drugs and that their comments about the products are factual and non-inflammatory.

Distributed Pharmaceutical Analysis Laboratory DRAFT validation manual 11/04/2014

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3. Limitations of the analytical methodology

Our procedures for quantifying the active ingredients in pharmaceutical dosage forms are based on monographs published in the United States Pharmacopeia (USP) or British Pharmacopeia (BP), but they are not pharmacopeia methods. Modifications must be made to the assay methodology to accommodate the circumstances of sample collection. For example, we analyze single drug tablets, rather than pooled samples of 20-50 tablets, simply because the samples available are single packs that do not include large numbers of tablets, and we may not have the level of quality assurance, maintenance, and record keeping in our HPLC facilities that a commercial lab must maintain.

In order to ensure that the strengths and limitations of the analytical methods are well understood by each user, we require two types of proof. First, before any pharmaceutical products can be assayed, the user must demonstrate the accuracy, precision, and linearity of their method and instrumentation according to standards laid out in USP <1226> and detailed below. We call this step method verification. Second, specific quality control samples must be run during the assay of pharmaceutical products. If these control samples do not assay correctly, as described in the directions for each analyte, the results from the pharmaceutical products must be thrown out and the samples re-run. We call this step quality control. Data from method verification and quality control must be submitted to the DPAL database to show proper performance of the method and assays


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4. Sample preparation

Sample storage and tracking:

The pharmaceuticals sent for analysis are forensic samples. They must be stored in a way that does not promote degradation or contamination, and you must keep good records of each sample so you can track who performed what tests on it and what the results were.

Samples must be stored in a cool, dark environment. We recommend a plastic bin with a snug lid in a refrigerator. Allow the sample to come to room temperature before you work with it, so water does not condense on the cold surfaces.

Each sample consists of at least 3 tablets or capsules which will be shipped in a plastic bag labeled with a UND tracking number. The tracking number will be of the form "14-xxxx" for a sample collected in 2014. When you run tests on individual tablets or capsules, label them as "14-xxxxa, b, c, etc" Have the students sign out samples for analysis and make sure they use the proper sample tracking number in their records and chromatogram labels.

External standards:

External calibration standards are created from analytical grade reagents that are traceable to USP or BP standards. The standard should include a certificate of analysis, and you must take the reagent purity into account when calculating its final concentration. Store dry standards as directed on the bottle (most must be kept cold).

amoxicillin: Sigma-Aldrich PHR1127-1g is traceable to USP and costs $51 per gram.

The "known" API standard should contain about 0.5 mg/ml of ampicillin, amoxicillin, or ciprofloxacin, and 0.5 mg/ml of amoxycillin plus 0.2 mg/ml of clavulanate if you're analyzing amoxi-clav. You will use this standard to determine all the other concentrations.

Calibration standards should span the range from 5% to 200% of the expected API concentration in the experimental samples and at least 5 standards should be used to construct the calibration curve. You will use these standards to establish linearity over the range of interest. It is OK to use the calibration curve if the samples are run at the same time as the unknown. A calibration curve generated on


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one day cannot be used to assay concentrations of samples run on another day. Since it takes 5 runs to do the calibration curve we prefer to establish linearity and then use a single-point external standard to assay concentrations of unknown.

Prepare a "normal" unknown sample in the 95-105% range, an "overdosed" sample in the 140-160% range, and a "deficient" sample in the 20-50% range. Also prepare a method blank which is nominally 0%.

Pharmaceutical dosage forms:

Samples for analysis should contain about 0.5 mg/ml of ampicillin, amoxicillin, or ciprofloxacin, and 0.5 mg/ml of amoxycillin plus 0.2 mg/ml of clavulanate if you're analyzing amoxi-clav. Accurately weigh a tablet or the contents of a gel capsule and take a portion of the powder that will give a 0.5 mg/ml solution when diluted to volume. Tablets should be crushed to fine powder in a mortar and pestle and well mixed, and contents of capsules should be well mixed. For preparation of the analytical sample, weigh out at least 25 mg of powder on an analytical balance; remaining powder should be labeled and frozen for storage.

For example, the total contents of an amoxicillin capsule with a nominal dose of 500 mg amoxicillin might weigh 627 mg due to excipients. To prepare a 0.5 mg/ml solution, a portion of roughly 63 mg would be accurately weighed and dissolved in 100 ml of solvent. Samples should be thoroughly mixed (eg by stirring on a magnetic stirrer for 5 min, or 5 min sonication) plus 2 min of hand shaking and inversion of the volumetric flask. All samples must be filtered through a fresh 0.45 micron syringe filter to remove particulates that might clog the HPLC column. Typically we will filter about 1 ml of the sample into an autosampler vial, discarding the first drops of filtrate.

Amoxicillin: Use 20 mM monobasic sodium phosphate at pH of 4.4 to make the samples. Keep standards and standard solutions refrigerated. Use standard solutions within 3 weeks. Alternatively, freeze for storage up to 2 months.

Ampicillin: Use 20 mM monobasic sodium phosphate at pH of 6.7 to make the samples. Keep standards and standard solutions refrigerated. Use standard solutions within 3 weeks. Alternatively, freeze for storage up to 2 months.

Amoxi-clav: Use DI water to make the samples. Clavulanate is thermally unstable and should be used within 6 hours of preparation.


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Ciprofloxacin: Use 25 mM phosphoric acid, adjusted to a pH value of 3, to make the samples. Keep standards and standard solutions refrigerated. Use standard solutions within 3 weeks. Alternatively, freeze for storage up to 2 months.

It would be a good mini-experiment to run periodic chromatograms for a standard sample that is left out at room temperature for several days.


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For ampicillin, amoxicillin and ciprofloxacin, the solution is made using the buffer used in the mobile phase. For amoxicillin we use For ampicillin we use 20 mM monobasic sodium phosphate at pH of 6.7. For ciprofloxacin we use 25 mM phosphoric acid, adjusted to pH of 3. For amoxicillin clavulanate the solution is made using water. Solutions are sonicated for around 5 minutes and then filtered using a 0.45 mircon syringe filter.

5. Method Validation

The goal is to establish that a standard USP or BP method, when run on your instrument and with your reagents, satisfies a set of analytical metrics. These directions are specific for HPLC methods using an external standard with a fixed-wavelength UV-Vis or diode-array detector.

Establish control chart: Set up a record keeping mechanism so you can track metrics such as the integrated intensity of the known standard for different analysts and days of operation. A page in the instrument log book can be used, or a paper chart, or a computer spreadsheet.

Precision: The relative standard deviation (RSD) for the integrated intensities of 6 injections of the known standard should be below 2%.

Linearity: Prepare and run at least five calibration standards over the concentration range of 5% to 200%. Calculate a regression line for the calibration data, including correlation coefficient, y-intercept, slope, and residual sum of squares. The correlation coefficient should be 0.98 or better and the y intercept should be zero within error of measurement.

Accuracy and range: Perform three replicate assays each of the overdosed sample, the normal sample, the deficient sample, and a solvent blank (total of 12 determinations). Run the external standard after every 5 runs and check that the integrated intensity for all the external standard runs falls within 2% RSD. Use the average external standard signal to determine the concentrations of the overdosed, normal, deficient, and blank samples. The measured concentration of each sample should be within 2 standard deviations of its true concentration.

Accuracy via spike recovery: If available, a sample of a pharmaceutical dosage form (tablet or capsule) of the target drug should be prepared for analysis and a portion spiked with an extra 30% of the API. Calculate the % recovery of the spike; it should be within 90-110%.

Specificity: This can be demonstrated by showing that a spike can accurately be recovered from a dosage form matrix (the test in the "accuracy via spike recovery" section). A more robust demonstration which provides a higher level of impurities and degradation products is to stress the dosage form (eg by baking the tablet or powder for an hour at 100C), then use that as the matrix for a spike recovery experiment.


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Optional: LOD and LLOQ: LOD or LLOQ determination is carried out using the slope of the

calibration curve and the SD of low concentration samples. Best practice is to prepare a sample at about 2-3 times the expected LOD; you could also use the SD for the blank runs. However you measure the LOD and LLOQ, samples near the LOD or LLOQ limit should be run and their chromatograms shown in the report.

6. Suggested instrument parameters, solvents, and gradients

a) AmoxicillinInstrument: Waters e2695 High Performance Liquid ChromatographColumn: Kinetex 250 x 4.6 mm C18 column, 5 μm particle size and 100 ÅTemperature: 30°CDetector: Waters 2998 Photodiode Array DetectorAnalytical Wavelength: 220 nm

Amoxicillin and Amoxicillin/clavulanate Method I:high flow rate, fast runs

Mobile Phase Buffer

Concentration of Buffer (mM)

pH of Buffer Sample Injection Volume (μL)

Monosodium Phosphate

20 4.4 20

Time (min) Methanol (%) Buffer (%) Flow (mL/min)

Ramp

0.0 5 95 1.5 None

7.0 5 95 1.5 None

7.1 90 10 1.5 Linear

8.5 5 95 1.5 Linear

10.0 5 95 1.5 NoneTable 1. Amoxicillin and Amoxicillin/Clavulanate Method I


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Time0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00

AU

0.0

5.0e-2

1.0e-1

1.5e-1

2.0e-1

2.5e-1

3.0e-1

3.5e-1

4.0e-1

13-0131 6-5-14 Sm (SG, 10x10) Diode Array 220

Range: 4.883e-1Area

3.4538724

2.4612102

Method 1 chromatogram (clavulanate at 2.46 min, amoxi at 3.45 min)

Amoxicillin and Amoxicillin-clavulanate Method II:lower flow rate, better if your column pressure is too high.

Mobile Phase Buffer




20 4.4 10


Ramp

0.0 5 95 0.5 None

1.0 5 95 0.5 None

6.0 80 20 0.5 Linear

8.0 80 20 0.5 Linear

13.0 5 95 0.5 Linear

18.0 5 95 0.5 None

Table 2. Amoxicillin or Amoxicillin/Clavulanate Method II

@@what are typical retention times?

Analytical metrics for amoxicillin:Column capacity factor 1.1-2.8 (if isocratic program is used),


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column efficiency >1700 theoretical plates,tailing factor <2.5 ,

RSD for replicate injections <2.0%.


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b) Ampicillin


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c) Amoxicillin-clavulanate (or amoxicillin-clavulanic acid)

Instrument: Waters e2695 High Performance Liquid ChromatographColumn: Kinetex 250 x 4.6 mm C18 column, 5 μm particle size and 100 ÅTemperature: 30°CDetector: Waters 2998 Photodiode Array DetectorAnalytical Wavelength: 220 nm

Amoxicillin and Amoxicillin/clavulanate Method I:high flow rate, fast runs

Mobile Phase Buffer




20 4.4 20


Ramp

0.0 5 95 1.5 None

7.0 5 95 1.5 None

7.1 90 10 1.5 Linear

8.5 5 95 1.5 Linear

10.0 5 95 1.5 None

Table 1. Amoxicillin and Amoxicillin/Clavulanate Method I


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Time0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00

AU

0.0

5.0e-2

1.0e-1

1.5e-1

2.0e-1

2.5e-1

3.0e-1

3.5e-1

4.0e-1

13-0131 6-5-14 Sm (SG, 10x10) Diode Array 220

Range: 4.883e-1Area

3.4538724

2.4612102

Method 1 chromatogram (clavulanate at 2.46 min, amoxi at 3.45 min)

Amoxicillin and Amoxicillin-clavulanate Method II:lower flow rate, better if your column pressure is too high.

Mobile Phase Buffer




20 4.4 10


Ramp

0.0 5 95 0.5 None

1.0 5 95 0.5 None

6.0 80 20 0.5 Linear

8.0 80 20 0.5 Linear

13.0 5 95 0.5 Linear

18.0 5 95 0.5 None

Table 2. Amoxicillin or Amoxicillin/Clavulanate Method II

@@ what are typical retention times?

Analytical metrics:


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resolution between the amoxicillin and clavulanate peaks must be at least 3.5, column efficiency for each peak at least 550 theoretical plates,

tailing factor below 1.5,RSD for replicate injections less than 2.0%.


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d) Ciprofloxacin

Instrument: Waters e2695 High Performance Liquid ChromatographColumn: Kinetex 250 x 4.6 mm C18 column, 5 μm particle size and 100 ÅTemperature: 30°CDetector: Waters 2998 Photodiode Array DetectorAnalytical Wavelength: 220 nm

Mobile Phase Buffer



Phosphoric acid 25 3 ???

Time (min) Buffer % Acetonitrile % Flow Rate (mL/min)0 95 5 13 70 30 15 70 30 17 95 5 110 95 5 1

retention times?

Analytical metrics:column efficiency >2500 theoretical plates,

tailing factor <4.0,RSD for replicate injections <1.5%


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7. Analytical metrics (how to tell if your HPLC is behaving properly):

Measuring theoretical plates: We recommend the British Pharmacopia method due to the simplicity of measuring peak width at 1/2 max height. tr is the retention time of the peak. This method will slightly underestimate column efficiency.

Measuring resolution of two peaks: Again, for peaks with tailing, it's easier to use a formula with peak widths measured at 1/2 max height:

Measuring tailing factor (USP method)

Measuring column capacity factor (for isocratic methods only): k'=(tr - t0)/t0, where tr is the peak retention time and t0 the dead volume of the column (measured by the elution time for the solvent front).


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8. Assay and Quality Control

External calibration standards are created from analytical grade reagents as directed in section 4. Five injections of the external standard must show a peak area within 2% relative standard deviation (RSD), and the range of retention times must be within 0.3 minutes. After every five unknown sample runs, the standard is injected as a quality check and it must assay within 2% RSD of the 5 initial injections and be within the 0.3 minute time range. If a quality check fails, data after the last passed quality check is not used.

Typically we analyze one pill from each package. If a sample fails analysis (assay value <90% or >120% of stated API content) then a new sample is prepared from the remaining powdered pill material and re-assayed (it would be good practice to perform this duplicate assay routinely). If this also fails, two more tablets are assayed.

A copy of this Excel spreadsheet is posted on the DPAL site.


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Version: This manual was drafted 11/4/2014 · Web viewFor ampicillin, amoxicillin and...

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Transcript of Version: This manual was drafted 11/4/2014 · Web viewFor ampicillin, amoxicillin and...