Qualification Of HPLC

In this slide contains definition and details of Qualification Of HPLC
Presented by: KHALID KUWAITY (Department of pharmaceutical analysis).RIPER, anantapur

1. QUALIFICATIN OF HPLC   A Seminar as a part of curricular requirement for Master of Pharmacy, I Year - Ⅱ semester Presented by KHALID OSMAN (20L81S0710) Department of Pharmaceutical analysis

2. Contents : Design Qualification Installation Qualification Operational Qualification Performance Qualification References 2

3. Introduction : 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: Design qualification(DQ) Installation qualification(IQ) Operational qualification(OQ) Performance qualification(PQ) 3

4. D e s i g n q ual i f i c a t i o n It describe the user requirements and defines the functional and operational specifications of the instrument. DQ should ensure that instrument to be purchased have the necessary functions and performance that will enable for suitable intended application. 4

5. Design qualification: Design element Example Maintenance •Vendor must deliver maintenance procedure and recommended schedule •Instrument must include early maintenance feedback for timely exchange of most important maintenance parts. •Maintenance procedure must be supplied on multimedia CD ROM Intended use Analysis of drug components and impurities. 5

6. Cont.… Design element Example User requirements specification for the HPLC analysis •Up to 100 samples/day. •Automated over night analysis. •Limit of quantitation:0.1%. •Automated confirmation of peak identity and purity with diode-array detection •Automated compound quantization and printing of report. FUNCTIONAL SPECIFICATION: •Pump •Detector •Auto sampler •Column compartment •Computer •Binary or higher gradient. •UV/VIS Diode array,190-900nm. •100 samples, 0.5µl to 5ml sample volume •15 to 60ºc controlled. •System control, data acquisition for signals and spectra, peak integration and quantitation 6

7. Cont.… Design element Example Operational specification •Detector: base line noise:5 x 10 -5 AU •Sampler: precision inj. Volume : <0.5%RSD. •Pump: precision of retain time: <0.5%RSD User instruction •Operation manual on paper •Computer based tutorial Qualification The vendor must provide procedures and services for IQ and OQ 7

8. Installation qualification : Installation qualification establishes that the instrument is received as designed and specified. It establishes that instrument is properly installed in the selected environment and this environment is suitable for the operation and use of the instrument. IQ should include analysis of a test sample. A successful run of such a sample verifies correct installation of all modules and electrical fluid connections. 8

9. Be f o re i n s t a ll a t i o n : Obtain manufacturers recommendations for installation site requirements. Check the site for the fulfilment of the m a nu f ac t u r e r s r ec o mm e n d a t i o n ( u t ili t i e s s u c h a s electricity and environment condition such as humidity and temperature). Allow sufficient shelf space for the equipment, SOPs, operating manual and software. 9

10. During installation : Compare equipment as received ,with purchase order (including software, accessories, spare parts). Check documentation for completeness (operating manuals, maintenance instruction, standard operating procedure for testing , safety and validation certificate) Check equipment for any damage. Install hardware (computer, equipment, fittings and tubing's, for fluid connection, column in HPLC, power cables, data flow and instrument control table). 10

11. Cont. ➢ Switch on the instrument and ensure that all modules power up and perform an electronic self test. ➢ Identify and a make a list with a description of all hardware, include drawings where necessary. ➢ Run test sample and compare chromatogram print out with reference chromatogram. ➢ Prepare an installation report. 11

12. Operational qualification ➢ It is the process of demonstrating that an instrument will function according to its operational specification in the selected environment . ➢ It verifies that the HPLC system compiles with key function and operational requirements as specified in the design qualification. ➢ In operational specification the supplier must define exactly the conditions that must be observed with varying conditions. E.g.: different ambient temperature. ➢ Before performing all other test first perform leak test if, it is failed then most of the remaining test will get failed. 12

13. T e s t p ara m e t e r s an d acc e p t anc e cri t e ria : Parameter Procedure User limit Lea k te s t i ng Flow test by volume or weight/time ±5% Baseline drift ASTM(American Society for Testing Material) Method < 2 x 1 -3 AU Baseline noise ASTM Method < 5 x 1 - 5 AU Precision of injection volume 6 x injection of caffeine standard, RSD of peak areas 0.3% RSD Precision of flow rate 6 x injection of caffeine standard, RSD of retention times 0.5% RSD Detector linearity Inject 5 standards < 1.5 A U , 5% R S D 13

14. Cont.… Parameter Procedure User limit W a v e l e n g t h A cc ur ac y Holmium oxide filter ± 1 nm Temperature Accuracy Co m p a r i s on w i t h e x te rn a l measuring device ± 1º c Temperature precision Monitoring temperature over 20 mins ±0.25 ºc Auto sampler carry over Injection of large sample after large concentration < 0.5% Mobile phase composition accuracy Step gradient from 4 to 7% B, Step heights relative to 100% w i t h acet one t r ace r ±1% 14

15. B AS E L I NE N O I SE AN D DR I F T 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. 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. The flow cells free from gas bubbles. To measure the drift of a UV detector, also make sure that all measuring conditions are constant. 15

16. 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. 16

17. 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 chrome Leon calculates a regression based on measured values , using the method of least square. The limit should be between <2 x 103 AU. To calculate the drift, chrome Leon calculates a regression line from all data points with in a range of 1- 21mins based on the method of least square. 17

18. Precision of injection volume: P r ec i s i o n o f i n j ec ti o n vo l u m e i s a n important pa r a m e t e r 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. 18

19. Detector linearity : L i n ea r i t y o f a de t ec t o r i s a c r i t i c a l pa r a m e t e r t o e s t ab li s h 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. 19

20. 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. 20

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22. Te m p e r a t ur e accuracy : Temperature fluctuations of the solvent and column can result in considerable retention time fluctuations. 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. 22

23. Cont.… The achieved temperatures are compared to the set values. The difference indicates the temperature accuracy and the limit should be in between ± 1 o C. 23

24. Au t o s a m p le r car r y ov e r : It is the appearance of an analyte in a run when a blank containing no analyte is injected. Evaluating auto sampler 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. It should be less than 0.5% . 24

25. Gradient mobile phase composition accuracy It is important for accurate quantitative analysis. Evaluating gradient mobile phase composition accuracy: An Acetone tracer is used to determine gradient mobile phase accuracy, stability and linearity. Make 6 compositions of water + acetone in concentration of 0%,20%, 40%,60%, 80% and 100% (20% increment). Linear ramp down from 100% to 0% is performed where the composition linearity is determined between ranges of 95,75 and 25%. 25

26. Cont.… All compositions accuracies are calculated as the absolute difference between the mean composition at each set point and the theoretical composition. 26

27. Performance qualification : Performance Qualification (PQ) is the process of de m o n s tr a t i n g t h a t a n i n s tr u m e n t co n s i s t e n t l y pe rf o r m s according to a specification appropriate for its routine use. P Q s h o u l d b e p e rf o r m e d o n a d a il y b as i s o r w h e n e v e r the instrument is used. The test frequency not only depends on the stability of the equipment but on everything in the system that may contribute to analysis the result for a liquid chromatography. 27

28. Cont.… The test criteria and the frequency should be determined during the development and the validation of the analytical method. In practice PQ mean system stability testing , where critical key system performance characteristic are measured and compared with documented , preset limit. For example, a well characterized standard can be injected 5 or 6 times and the standard deviation of amounts are then compared with predefined value. 28

29. Cont.… If the limits of detection and quantifications are critical, the lamps intensity profile or the base line should be tested they should use the same column and chemicals for the real sample. 29

30. References : 1. P.Bedson and M.Sargent , The development and application of guidance on equipment qualification of analytical instruments, Accreditation and Quality Assurance, 1 (6), 265/274 (1996) . L. Huber, Validation and qualification in analytical laboratories, Text book, 300 pages, 1998, Interpharm , Bufallo Grove, IL, USA, Nov. 1998 http://www.t-online.de/home/HUBERL/validat.htm (book) . L. Huber, Equipment Qualification in Practice LC/GC Magazine, Vol 16, February 1998, 148-156 http://www.t-online.de/home/HUBERL/validat.htm (book). 30

31. Thank you 31