LAB-090 HPLC Reproducibility, Column Performance and Testing Guidelines

Reproducibility tests or Calibration tests as also known, are carried out on each HPLC system as a measure the system conformance prior to running assay tests on the system.

Column performance tests are carried out on each column to be used in a HPLC method as a measure of Column conformance prior to assay determinations.

General HPLC testing guidelines have been included in this SOP.

1. Reproducibility (Calibration) Tests

Reproducibility tests must be carried out on each HPLC system on a weekly basis, or if the system is used irregularly, prior to system use.  When in constant use, calibrate weekly. Reproducibility tests must also be carried out if the system has been repaired or has been moved to another facility.  (Note that Validation must also be carried out if the system is moved- Refer to Supplier’s notes).  Confirm calibration status by referring to “Calibration folder” for the HPLC system to be used.

The “Calibration” test is based on the calculated percent Relative Standard Deviation in Retention time and Peak area resulting from a designated minimum number of six standard injections. The calibration technique is left at the discretion of the Laboratory technician.

Calibration can be done using any HPLC method.  Set up the HPLC instrument to run a chosen method, and consider the following variables when choosing the calibration techniques:

a. Reference standard substance stability

b. Standard solution stability over run time period

c. Volatility of standard solution being injected

d. Injection volume

e. Environmental effects, eg air conditioning

f. Any other variables relevant which may not be mentioned here

Calibration techniques

a. Six consecutive standard injections from the 1 vial or

b. Six consecutive standard injections from 6 different vials or

c. Six standard injections throughout run

Note: All injections chosen must be from the one HPLC system and run on the same day. It is preferred that a standard solution is used for the calibration, but a homogenous sample may also be used.

The resulting Retention times and Peak areas are used in the following calculations (Refer 1.1 “Reproducibility calculations for Peak area and Retention times”) to calculate Reproducibility.

Refer section 1.4 “Recording results” for details on record keeping requirements.

1.1. Reproducibility Calculations for Peak Area and Retention Times:

Mean, X  =  ∑ Yı

                      n

Standard Deviation, S = √∑ (Yı –X ) ²

                                             (n – 1)

Coefficient of variation, V  = S  x 100 

                                                        X

also known as % Relative standard deviation (RSD)

Where,

Yı = Individual values of Peak area or Retention time
n = Number of injections

Empower offers a reporting option to calculate chromatograph summaries, where these calculations are done automatically through the Report option.

1.2. Expected value

The Relative Standard Deviation for Peak Areas should be less than 2%.  The Relative Standard Deviation for the Retention Times should be less than 1%.

1.3. Detector Checks

The power supply and intensity of the Detector lamp play important roles in determining the intensity of signal output which in turn affects the peak area recorded, and thus the calculated concentration of sample.  Consult the various manuals in order to check the recommended outputs for power supply and lamp intensity.

The test methods may have the provision to use Photodiode array detectors (PDA), Fluorescence detectors, Electrochemical detectors (ECD), Refractive index detector (RI), and Evaporative Light Scattering detector (ELSD).

The PDA detector is checked for conformance using an internally designed Empower diagnostics method.  To run the diagnostics test, from the Quickset screen, choose the PDA diagnostics option, select all tests and run.

The Waters 470 Scanning Fluorescence detector is controlled by monitoring the powering up displays and the hours lamp is used.

The RI is checked to ensure its temperature is 35°C and has been autozeroed

The ECD is controlled by checking that working electrode has been cleaned and the reference electrode solution changed 6 monthly

The ELSD is checked by running the diagnostic tests for the Optics, Nebuliser and Flow meter.

See section 1.4 “Recording results” for details on record keeping requirements.

1.4. Recording results

The “Reproducibility” results and the “Detector checks” results are to be transferred onto the appropriate “Calibration of HPLC” form (Form-720 HPLC Records, page 1 or 2). Compile a print out of the Empower Calibration summary report for the standard injected and file together with the completed form, in the “Calibration records” folder for the specific HPLC system.  Complete also the “HPLC Calibration Log” (Form-720 HPLC Records, page 3) kept at the beginning of the “Calibration records” folder.

For the HPLC systems used on a regular basis by the same Laboratory Technicians, it is the responsibility of these Laboratory Technicians to ensure that the form “HPLC Calibration Log” (Form-090 HPLC Records, page 3) is completed on a weekly basis, even if calibration was not done on the system.  This would assist with the monthly “House keeping audit” follow through.

Note:

If the HPLC is found to be out of Calibration follow in order the following steps:

(a) Record an explanation of problem on the “HPLC Calibration Log” (Form-720 HPLC Records, page 3).

(b) If the fault can be singled out as user error, or is rectified simply (eg. Air bubble in detector), correct the fault and then re-do the Calibration/ Detector check, fill out a second “Calibration of HPLC” form (Form-720 HPLC Records, page 1 or 2) and attach to first form.  Confirm Calibration by adding entry onto “HPLC Calibration Log” (Form-720 HPLC Records, page 3).

(c) If the fault cannot be singled out, refer concerns to HPLC service person for repair or advice.  If the instrument will need repair, place an “Out of Calibration – Do not use” label on the HPLC.

(d) Inform Laboratory Supervisor.

(e) Ensure the HPLC is repaired.  File the completed service report in the “Service & Repair” folder for that system/s.

(f) Re-confirm Calibration of the HPLC by repeating section 1 of this SOP.

(g) Remove the “Out of calibration – Do not use“ label from the HPLC.

2. Column Performance Tests

Column performance tests involve monitoring the changes in Efficiency (N), Capacity (K’) and USP tailing characteristics of the specific column over time.  This provides valuable information about the state of the column, and whether it can or cannot be used in further analysis.

Columns are generally dedicated to run the same HPLC testing method for a specific product. This provides better control of the column history.  Initially, the column is tested with HPLC methods pre-determined by the manufacturer of the columns.  (See Table 2.5 “Supplier’s Column performance methods for Columns and Cartridges”.)  With time the performance is monitored using the testing method for the product tested with the specific column.

The peaks resulting from the standard solution injection onto the system are used in calculation to determine the performance of the column.  (Refer to section 2.2, 2.3, & 2.4 for detailed explanation of tested parameters).  These parameters are calculated by Empower on a regular basis and can be extracted by choosing the option “5 sigma” or “N(Half Height)” (Efficiency), “K prime” (Capacity factor), “Resolution” (Resolution), & “USP tail“ (Tailing factor) when printing out the report.

2.1. Frequency of Column Performance Tests

a) Test New Column with Manufacturer’s recommended method (see Table 2.5 “Supplier’s Column Performance methods for Columns and Cartridges”).  Print out resulting chromatogram and record the results onto “Record of performance tests for HPLC Columns” (Form-720 HPLC Records, page 4)   Label the Column with a tag that indicates the serial number of the Column, the Product the Column has been dedicated to, and the date the Column was first used.

b) Compare the resulting information from test above “a)” to the Manufacturer’s Column performance certificate, if available.  Significant deviations in performance should be referred to the Supplier.

c) From the first HPLC test method done with the Column, print out of a Standard injection chromatogram to file in “Column performance” folder for the specific HPLC systems, and complete “Record of performance tests for HPLC Columns” (Form-720 HPLC Records, page 4)

d) Every month, or every time test method is used on the Column (for more remotely used methods), repeat step c)

e) For Methods which use a pre-column as well, do not do steps “c)” & “d)”, instead repeat manufacturer’s recommended method, step “a)” every 6 months.  The pre-column will interfere with the true resulting information for the column used.

f) The Laboratory Technician must monitor column performance.  If the Column Performance monitoring factors (N, k’, USP tailing) show a significant reduction in performance, the Laboratory Technician must use their discretion and restore the column, (refer to supplier information) or discard the column.

If a column is discarded, make a note to indicate so on the form “Record of performance tests for HPLC Columns” (Form-720 HPLC Records, page 4).

Note: When removing the column from the HPLC system, cap the column tightly on both ends, to avoid column drying up.

2.2. Column Efficiency (Plate count)

There are several ways of calculating efficiency (eg Five Sigma, Four Sigma, Half Height).  Five Sigma is the preferred method because Five Sigma values reduce markedly when irregularities in peak shape occur.

Five Sigma is defined in terms of the number of theoretical plates (N) per column and is calculated using the equation:

N (Five Sigma)= 25 x  VR ²

                                      W²

Where,

VR =  the distance along the baseline between the point of injection and a perpendicular dropped from the maximum of the peak of interest.

W =   the width of the peak of interest at 4.4% of peak height, measured in the same units as VR (see below)

Figure 1: Column Efficiency

When two peaks elute very close together five Sigma cannot be calculated.  In these cases, Half Height (which is also defined in terms of the number of theoretical plates, N, per column) can be used to calculate the column efficiency.

         N (Half Ht) = 5.54 x  VR²

                                             W²

Where,

VR = the distance along the baseline between the point of injection and a perpendicular dropped from the maximum of the peak of interest.

W = the width of the peak of interest at 50% of peak height, measured in the same units as VR.

2.3. Capacity Factor

Capacity Factor, (k´) is defined by the equation:

k´  =        V_R  ‑  V_o
               ———–

                     V_o

Figure 2: Capacity Factor

V_R   =   the distance along the baseline between the point of injection and a perpendicular dropped from the maximum of the sample peak.

V_o   =    the distance along the baseline between the point of injection and a perpendicular dropped from the maximum of an un-retained peak.

To optimise separations, k´ values between 1 and 5 are recommended for the peak of interest.
k´ values less than 1 may compromise specificity whereas values greater than 5 will lead to long analysis times.

2.4. USP Tailing

The Tailing factor, T is an indicator of peak skewness and is calculated using the equation:

T =    W

         2F

Figure 3: USP Tailing

Where:

W = Peak width at 5% of peak height

F = Distance between the perpendicular dropped from the peak maximum and the leading edge of the peak at 5% of the peak height.

2.5. Efficiencies and Column Performance methods for Columns and Cartridges

The Efficiencies in the table below are a guide as to what to expect when a column is new and is tested using the Column performance tests listed below:

Table 2.5

Approximately 2µL of sample should be injected, unless otherwise stated. (Ensure the injection volume used is the same as previously used to compare same column types).

Phosphate Buffer, pH 7.0:

Mix 7.5mL of IM Sodium dihydrogen phosphate with 28.5mL of 0.5M Disodium hydrogen phosphate and dilute to 1000mL.  Adjust the pH to 7.0 with ortho Phosphoric acid.

Racemic adrenaline

20µg/mL (+/ -)adrenaline (10µg/mL of l-form and 10µg/mL of d-form)

Excipient solution

50 mg Sodium metabisulphite and 0.56g NaCl dissolved in 100mL Distilled water.  Adjust pH to 4 with HCl.

Acetophenone, Uracil, and Toluene Solution

Weigh 30 mg of each of Acetophenone and Uracil into a 100 ml volumetric flask.  Add 3 grams of Toluene.  Dissolve and make up to volume with MeOH.

3. Testing Analysis

It is recommended that when doing assay analysis for regular production/at release testing, stability testing and trial stability, that the procedure is uniform.  The procedure may be varied at the discretion of the Laboratory Technicians where variables as mentioned in section 1 may be applicable.

Following is a guide to set up sample set test runs using HPLC:

Note:
The Standard is the prepared reference to be tested against, and the Sample is the product as prepared for analysis.

3.1  At Release/Production & Stability samples

Inject Standard solution, and then four Sample solutions.  Where a second or more standards are used, as in degradation, inject the Standards consecutively and then the four Samples.  Each batch of product must be injected at least once.

3.2 Trial stability samples

Inject Standard solution, and then four Sample solutions.  Where a second or more Standards are used, inject the Standards consecutively and then the four Samples.  Each batch of product must be injected at least twice, and the results calculated and recorded individually for each of the two injections.

4. Summary of Changes