Qualification of HPLC

University Institute of Pharmaceutical Sciences is a flag bearer of excellence in Pharmaceutical education and research in the country. Here is another initiative to make study material available to everyone worldwide. Based on the new PCI guidelines and syllabus here we have a presentation dealing with qualifications of HPLC which is the " High Performance Liquid Chromatography".
Thank you for reading.
Hope it was of help to you.
UIPS,PU team

1. QUALIFICATION OF HPLC PRESENTED BY:- MANGESH LAVANGE Guide- dr. indu pal kaur M PHARM 1 ST SEM, PA UIPs, Panjab university Chandigarh, 160014

2. What is Qualification?  Action of proving and documenting that equipment or ancillary systems are properly installed, work correctly, and actually lead to the expected results. The entire qualification consists of four parts: 1. Design qualification(DQ) 2. Installation qualification(IQ) 3. Operational qualification(OQ) 4. Performance qualification(PQ).

3. Types of qualification Level I Level II Level III Level IV Design Qualification(DQ) Installation Qualification (IQ) Operational Qualification (OQ) Performance Qualification (PQ)

4. High performance liquid chromatography ( HPLC ) HPLC is a form of column chromatography that pumps at high pressure a sample (analyte) dissolved in a solvent (mobile phase) through a column with an immobilized chromatographic packing material (stationary phase). As the sample passes through the column, analytes having the strongest interactions with the stationary phase exit the column the slowest, meaning they exhibit the longest retention times. In contrast, samples demonstrating little interaction with the column material elute quickly and are thus characterized by short retention times. The properties of the sample and the solvent, as well as the nature of the stationary phase, determine the retention time of the analytes, or how fast they pass through the column. Upon exiting the column, the mobile phase passes through a detection module, such as a fluorimeter or a UV-absorbance detector. Selection of the appropriate detector and monitoring wavelengths is essential for optimizing the sensitivity of HPLC detection.

5. Qualification of HPLC Qualification of HPLC involves 4 levels. The core document “Qualification of Equipment” contains the common information of level 1 and level II qualification which is common to all types of instruments. Level III and level IV qualifications must be carried out being an ISO17025 requirement. Combined test procedures can be applied to carry out Level III and Level IV qualifications to check several parameters simultaneously. E.g. overall system performance test for peak area precision, retention time precision, gradient reproducibility, etc.

6. Continued… Level I Qualification : At level I of the qualification of a HPLC ( selection of instruments and suppliers) it is recommended to select a manufacturer of HPLC instrument that can satisfy the needs of the laboratory and works under ISO 9001 certification. Level II Qualification : At level II of the qualification of a HPLC instrument ( installation and release for use ) it is recommended to check all requirements set during the selection of the instrument, and calibration should be performed before putting into service by an accredited external service supplier, or internally y appropriately qualified personnel, using certified reference buffers according to an approved procedure. Level III Qualification : Periodic and motivated instrument checks. Various recommendations and acceptance limits are qualified in this level of qualification.

7. Instrument module Parameter to be checked Typical acceptance limits Solvent delivery system Flow rate accuracy(HPLC) Flow rate accuracy (UHPLC) Flow rate precision ( HPLC and UHPLC) Gradient composition accuracy Gradient ripple ± 5.0% ± 3.0% RSD ≤ 0.5% ± 2.0% ≤ 0.2% Injector Volume precision (HPLC and UHPLC) Carry-over RSD ≤ 1.0% ≤ 0.2% Autosampler Thermostating accuracy ± 3˚C Oven or cooling device (column) Thermostating accuracy Thermostating stability ± 2˚C ≤ 1˚C Multi-wavelength detector Linearity Wavelength accuracy Drift r² ≥ 0.9990 ± 2nm Annex I

8. Fluorescence detector Wavelength accuracy ( excitation and emission) Signal/Noise ratio ± 3nm ≥400 Electrochemical detectors Amperometric detection Integrated amperometric detection Conductivity detection Coulometric detection Drift (cell current) Noise Linearity Drift Noise Linearity Drift Noise Annexure 1 ≤8 pA/h ≤2 pA r² ≥ 0.9990 ≤1250 pC /20 min MW 20 readouts <160 pC r² ≥ 0.9990 ≤2 nS ≤20 nS /h Annexure 1 Refractive index detector Signal/noise ratio Drift over time linearity ≥10 ±0.1 mV/min r² ≥ 0.9950

9. Circular dichroism detector Linearity Signal/noise ratio Drift over time Spectra comparison r² ≥ 0.9950 >1.0 ≤0.1 meg/h ± 4nm Charged aerosol detector Baseline noise Largest random spike Baseline drift Repetablility Signal/ noise ratio Signal calibration ≤0.04 pA ≤0.2 pA ≤0.04 pA/min RSD ≤ 10% ≥10 r² ≥ 0.9990 Evaporative light scattering detector Noise Baseline drift Repeatability ≤2 mV ≤2.0 mV/h RSD ≤ 3.0%

10. Level IV Qualification In-use instrument checks Parameters to be checked Acceptance limits System suitability According to validated in-house method Peak area precision (assay, application to the main peak of the analyte when not saturated) Peak area precision related substances RSD ≤1.5% (minimum 5 injections of test or reference solution) (unless otherwise prescribed in the system suitability of the method e.g.: specific requirements dossiers) RSD ≤5.0% (minimum 3 injection of the diluted solution or reference solution used for quantification) (unless otherwise prescribed in the system suitability of the method e.g.: specific requirements)

11. Retention time precision (applicable to the main peak of the standard solution when not saturated) RSD ≤ 2.0% (minimum 5 injections of test or reference solution) Carry-over (by comparing consecutive injections of a standard solution of the substance being quantified and a blank injection) ≤0.2% (assay) Below disregard limit (related substances) Signal/ noise ratio ( to be applied for related substances test only)

12. BASELINE NOISE AND DRIFT: •Drift and baseline noise are important factors for UV detectors. Increased baseline noise considerably reduces the sensitivity, as it is not possible to distinguish between low-level signals and noise. With increased drift, it is more difficult to integrate the signals correctly because the less stable the baseline is, the more inaccurate is integration. •The baseline noise of the detector mainly depends on the lamp. There is a considerable increase in noise if an old lamp with poor light intensity is used. This is also true when the flow cells is dirty. In addition make sure that the flow cells free from gas bubbles. •To measure the drift of a UV detector, also make sure that all measuring conditions are constant. In addition, it is very important that the lamp has been burning for several hours in the detector environment, avoid direct sunlight. •The lamp intensity decreases while the lamp is burning. Besides, the lamp ages when it is turned on and off very often.

13. Continued… EVALUATING BASELINE NOISE AND DRIFT: •TO check noise, drift water is pumped through the cell at a flow rate of 1ml/min. The UV signal is recorded at 254nm. •To calculate noise the measuring signal is split into 20 intervals for 1min each. For each interval chromeleon calculates a regression based on measured values, using the method of least square. The limit should be between <2 x 10- 3 AU. •To calculate the drift, chromeleon calculates a regression line from all data points with in a range of 1-21mins based on the method of least square. The slope of the regression line is the calculated drift. The limit should be between <5 x 10—5 AU.

14. PRECISION OF INJECTION VOLUME: •Precision of injection volume is an important parameter for accuracy of quantitation. EVALUATING PRECISION OF INJECTION VOLUME •Inject 6 standard caffeine solution and calculate height, area, average height, average area, %RSD of height and %RSD of area which gives precision of volume and the limit should be in between 0.3% RSD. DETECTOR LINEARITY: •Linearity of a detector is a critical parameter to establish for reliable and accurate quantitative results. EVALUATING DETECTOR LINEARITY : •A series of 5 traceable standards (caffeine solution of concentration about 0.00035 to 0.35mg/ml) are injected and evaluated. The detector linearity is calculated by determining the peak area vs concentration. %RSD can also be calculated for checking the detector linearity. The limit should be in between >1.5 AU, 5% RSD.

15. Wavelength accuracy:  •It is an important parameter for accuracy of quantitative and qualitative analysis. EVALUATING WAVELENGTH ACCURACY: •Traceable caffeine standard is used to determine the wavelength accuracy. Caffeine is trapped in the flow cell and a programmable timetable is used to determine the wavelength maxima (205nm) and minima (273nm). The wavelength accuracy is determined as the absolute difference between the measured and certified wavelength values. TEMPERATURE ACCURACY: •Temperature fluctuations of the solvent and column can result in considerable retention time fluctuations. Therefore, accuracy of the temperature is important. EVALUATING TEMPERATURE ACCURACY : •4 measuring points are used to check the temperature accuracy of the column compartment. The check is performed with column oven sequence. The achieved temperature is measured with external calibrated thermometer. •The achieved temperatures are compared to the set values. The difference indicates the temperature accuracy and the limit should be in between ± 1º c 

16. Temperature precision: Monitor temperature for 20 min. and limit should be in between ±0.25˚C. Auto sampler carry-over After a highly concentrated sample, a sample containing only solvent is injected. Ideally only the signal for the solvent is displayed in the chromatogram. However, if a signal for the sample is displayed, this indicates the carry over by the autosampler. EVALUATING AUTOSAMPLER CARRY OVER: Run the sample containing only solvent. The signal for solvent will be displayed . If other signals are displayed it is due to auto sample carry over. Should be less than 0.5%

17. Documentation: On completion of equipment qualification , documentation should be available that consists of : Design qualification document . IQ document (includes description of hardware and software) . Procedures for OQ testing . OQ test reports ( includes test parameters, acceptance criteria and actual results. PQ test procedures and representative results.

18. References: Ph.Eur.2.2.35 chromatography; High performance liquid chromatography Guidance on equipment qualification of analytical instruments Journal of Perkin Elmer life & analytical science https://www.edqm.eu/sites/default/files/quality_management_document_annex_1_qualification_of_hplc_equipment.pdf