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1. Dr. Rajnikant B. Mardia Senior Assistant Professo r Department of Pharmaceutical Chemistry Kamlesh Jivani M.Pharm sem – II ID No. 21MQPOS005 Dept. Pharmaceutical Quality assurance Faculty of Pharmacy, DDU, Nadiad Guided by, Prepared by, QUALIFICATION OF HPLC SYSTEM AND LC-MS SYSTEM

2. 2 QUALIFICATION OF HPLC SYSTEM

3. Introduction Equipment qualification is a formal process that provides documented evidence that an instrument is fit for its intended use. The entire qualification process consists of four parts: Design qualification (DQ) Installation qualification (IQ) Operational qualification (OQ) Performance qualification (PQ) 3

4. Design qualification (DQ) Design qualification (DQ) describes the user requirements and defines the functional and operational specifications of the instrument. Design elements Examples Intended use Analysis of drug compounds and impurities User requirement specification for the HPLC analysis 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 quantitation and printing of report 4

5. Design elements Examples Functional specifications Autosampler - 100 samples, 0.5 µl to 5 ml sample volume Column compartment - 15 to 60˚C, peltier controlled Detector - UV/Vis Diode-array, 190-900 nm Computer - System control, data acquisition for signals and spectra, peak integration and quantitation, spectral evaluation for peak purity and compound confirmation. Operational specifications Detector: Baseline noise < 5 X 10 -5 AU Sampler: Precision injection Volume sample carry over: <0.5% Maintenance Vendor must deliver maintenance procedure and recommend schedule Training Vendor must provide familiarization and training 5

6. Installation qualification (IQ) Before installation Check the site for the fulfillment of the manufacturer’s recommendations (utilities such as electricity, and environmental conditions such as humidity and temperature). Allow sufficient shelf space for the equipment, SOPs, operating manuals and software. During installation Compare equipment as received, with purchase order (including software, accessories, spare parts) Check documentation for completeness (operating manuals, maintenance instructions, standard operating procedures for testing, safety and validation certificates) 6

7. Check equipment for any damage Install hardware (computer, equipment, fittings and tubings for fluid connections, columns in HPLC, power cables, and instrument control cables) Switch on the instruments and ensure that all modules power up and perform an electronic self-test. Run test sample and compare chromatogram with reference chromatogram Prepare an installation report Installation qualification (IQ) 7

8. Performance qualification (PQ) Parameter Procedure User Limit Leak testing Flow test by volume or weight/time ± 5 % Baseline drift ASTM Method <2 × 10-3 AU Baseline noise ASTM Method <5 x 10-5 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 8 ASTM Method → American Society for Testing and Material Method

9. Parameter Procedure User Limit Detector linearity Inject 5 standards (caffeine solution) >1.5 AU, 5% RSD Temperature accuracy comparison with external measuring device ± 1 °C Temperature precision monitoring temperature over 20 min ± 0.25 °C Auto sampler carry over Injection of blank solvent after large concentration < 0.5 % Mobile phase composition accuracy Step gradients from 4 to 7 % B, step heights relative to 100%, with acetone tracer ± 1 % 9

10. BASELINE NOISE AND DRIFT W ater is pumped at a flow rate of 1ml/min UV signal is recorded at 254nm To calculate noise the measuring signal is split into 20 intervals for 1 min each. For each interval chromeleon calculates a regression based on measured values, using the method of least square, then determine maximum distance of two data points above and below the line. L imit should be between <2 x 10 -3 AU 10

11. 11 Figure: Long-term noise measurement Figure: Short-term noise measurement

12. For drift Chromeleon calculates a regression line from all data points with in a based on the method of least square. The slope of the straight line is expressed in absorbance units/hrs can be provide an estimation of detector drift. Limit should be between <5 x 10—5 AU. 12 Figure: Drift measurement

13. DETECTOR LINEARITY A series of 5 traceable standards (caffeine solution of concentration about 0.00035 to 0.35mg/ml) are injected. The detector linearity is calculated by determining the peak area vs concentration. %RSD can also be calculated for checking the detector linearity. limit should be in between >1.5 AU, 5% RSD. 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. WAVELENGTH ACCURACY 13

14. TEMPERATURE ACCURACY M easuring 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 ± 1ºc TEMPERATURE PRECISION Monitor temperature for 20 minutes and limit should be in between ±0.25 º 14

15. 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. It Should be less than 0.5% 15

16. GRADIENT MOBILE PHASE COMPOSITION ACCURACY It is important for accurate quantitative analysis.  Acetone tracer is used to determine gradient mobile phase accuracy, stability and linearity. Make 6 compositions of methanol and acetone or caffeine 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%.  All compositions accuracies are calculated as the absolute difference between the mean composition at each set point and the theoretical composition. 16

17. 17 Solnent channel A – Methanol Solvent channel B - Caffeine

18. QUALIFICATION OF LC-MS SYSTEM 18

19. The day-to-day performance of LC-MS or LC-MS/MS depends on its calibration, tuning, system suitability test, and final overall validation. Calibration Parameters Calibration parameters are instrument parameters whose values do not vary with the type of experiment such as, Peak width Peak shape Mass assignment Resolution versus sensitivity 19

20. 1. Peak width Peak width depends on the mass resolution. A resolution of 1 mass unit is sufficient to distinguish ions in most qualitative/quantitative small molecule applications. Unit resolution → when the peak width at half-height is about 0.6 to 0.8 mass unit. The profile scan of ions on a typical benchtop LC-MS has a bandwidth of about 1 mass unit (Figure 1). Figure 1: Band width in a typical benchtop LC-MS 20

21. 2. Peak Shape and Profile Scan In a typical benchtop LC-MS, abundance measurements are collected at 0.10-m/z increments. When these data are presented in a mass spectrum, a single line can be shown. The height and position are derived from the profile scan. 21

22. 3. Mass Assignment It is performed using specific MS calibrants. Calibrants should be well-characterized reference materials. LC-MS qualitative analysis → Use reference materials LC-MS quantitative analysis → Use a combination of reference materials and actual analyte standards as calibrants. 4. Resolution versus Sensitivity Mass resolution is a compromise between ion intensity and peak width. In general, as the resolution is increased, the ion intensity decreases. 22

23. Calibration Comparison of a measurement standard or instrument of known accuracy with another standard or instrument to eliminate deviations by adjustment. Manual, semiautomatic, and automatic LC-MS calibration require introduction of the solution of the calibrant (calibration solution) into the MS at a steady rate while the procedure is running. Calibration solution is introduced directly (infused) into the MS from a syringe pump or through a loop injector connected to the LC pump. MS must be calibrated at least once every three months . 23

24. Tuning Parameters Tuning parameters are instrument parameters whose values can vary with the type of experiment. Manual, semiautomatic, and automatic tuning procedures require the introduction of a tuning solution of the analyte of interest into the MS at a steady rate. This can be done in following three different ways: 1. By introducing the solution directly from the syringe pump (direct infusion) → This method is good for tuning for experiments at a low flow rate. 2. By introducing the sample from the syringe pump into the effluent of the LC by using a tee union 3. By injecting the sample into the effluent of the LC by using a loop injection valve [flow injection analysis (FIA)] → second and third methods are useful for experiments at a higher flow rate. 24

25. Optimized parameter which affect the signal quality, change from instrument model to instrument model & from brand to brand. Examples are Source, temperature, ionization voltages, gases (nebulization, desolvation, and collision), ion path potentials (lens, multipoles, or stacked rings), collision energy, solution, or mobile-phase flow rate. The potentials, RF values, and gas pressure affect the declustering, focusing, fragmentation, and efficiency of ion transmission. 25

26. System Suitability Testing Tests allows the determination of system performance by analysis of a defined solution prior to running the analytical batch. System suitability should test the - Entire analytical system - Chromatographic performance - Sensitivity of the mass spectrometer for the compounds of interest . 26

27. Validation Final step to guarantee that a LC-MS system performs as expected. Validation includes instrument calibration, tuning, testing, and checking of the documentation for completeness, correctness, and compliance with SOPs. Validation consists of four separate steps: Validation of the instrument and the computer controlling it (computer system validation or CSV) Validation of the analytical method running on that equipment System suitability testing, to test the equipment and the method together to confirm expected performance QA/QC review of sample analysis data collected on such a system 27

28. MS CALIBRATION PARAMETER This is the window used by the calibration software to search for the most intense peak. Increasing this window gives a greater chance of incorrect peak matching. It is important to ensure that the correct peak is located in the peak search range; otherwise, a deviation in the calibration may arise. If an incorrect peak is located, the search peak range should be adjusted to locate only the correct ion. 28 1. Peak search range

29. 2. Peak threshold All peaks in the acquired spectrum below the intensity threshold value (measured usually as a percentage of the most intense peak in the spectrum) will not be used in the calibration procedure. 3. Peak Width This is a measure used to set the resolution, usually specified at 50% of maximum intensity. 29

30. Calibration solution Calibrants are required to calibrate the mass scale of any mass spectrometer. Calibrants commonly used in electron ionization (EI) and chemical ionization (CI), such as perfluorocarbons , are not applicable in the ESI mode. The main calibrants used or still in use to calibrate ESI-MS can be divided into the following categories: polymers, perfluoroalkyl triazines, proteins, alkali metal salt clusters, poly ethers, water clusters, and acetate salts. 30

31. 1. Polymers polypropylene glycols (PPGs) and polyethylene glycols (PEGs) are the preferred calibrant for many small molecule applications. PPG calibration solutions produce mostly singly charged ions over the entire instrument mass range in both positive and negative ion mode. The PPG ions in general used for calibration in positive mode are: 59.0 (solvent and fragment ion), 175.1 (fragment ion), 616.5, 906.7, 1254.9, 1545.1, 2010.5, and 2242.6 31

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33. 2. Perfluoroalkyl Triazines Perfluoroalkyl triazines such as Ultramark 1621 have mainly been used as the calibrant for FAB and ESI-MS calibration. This standard is very sticky and is very difficult to remove from the ion source. For this reason, Ultramark 1621 calibration solution should not be used at flow rates above 10 µL/min, to avoid system contamination. Ultramark 1621 is in general used together with other calibrants to cover the entire MS range. Calibration solutions containing caffeine , L-methionyl- arginyol - phenylalanylalanine acetate H2O (MRFA) and Ultramark 1621 are commonly employed for ESI-MS calibration. 33

34. 34 caffeine: m/z 195; MRFA: m/z 524; and Ultramark 1621: m/z 1022, 1122, 1222, 1322, 1422, 1522, 1622, 1722, 1822.

35. 3. Proteins ESI-MS calibration was initially performed using solutions of gramicidin S, cytochrome c, or myoglobin as calibrant. Proteins produce multiply charged species in ESI. When proteins are used as calibrants for low-resolution LC-MS and LC-MS/MS instruments, it is not possible to resolve individual isotope peaks. 35

36. 4. Alkali Metal Salt Clusters It cover a wide m/z range and are used to calibrate mass spectrometers in ESI mode. Cesium iodide solutions produce singly and doubly charged species from m/z 133 up to m/z 3510 or higher. Cesium iodide calibration solutions are not very commonly used due to the following drawbacks: sample suppression persistence in the ESI source possible cation attachment 4. The large spacing 260 amu between peaks. 36

37. A mixture of sodium iodide and rubidium iodide calibration solution is able to cover the instrument’s full mass range from 23 to 3920. The peak at 23 is sodium, the 85 peak is rubidium, and the others are clusters. 37

38. 5. Polyethers Polyethers such as polyethylene oxide (PEO) and polypropylene oxide (PPO) have been used for ESI-MS calibration. sodium attachment is frequently observed, due to traces of sodium in solvents and glassware. Macrocyclic polyethers are also used as ESI-MS calibration. Derivatized polyethers such as polyether sulfate have been investigated for both positive and negative-ion calibration. Lauryl sulfate ethoxy- lates were also used as calibrants for negative-ion ESI. Polyether amines and quaternary ammonium salts were used as positive-ion calibration solutions. 38

39. 39 6. Acetate salts Sodium acetate and sodium trifluoracetate clusters were used and produce useful reference peaks for both positive and negative ESI. 0.5% acetic acid in ammonium acetate solutions can be used for calibration in ESI-MS.

40. Reference Chung Chow Chan et al, Analytical method validation and instrument performance verification, 2004, page no.173-184 and 197-216. Dr. Ludwig Huber qualification of high performance liquid chromatography system, Bio pharm journal, vol 11 page number 1 to 9. 40

41. T H A N K Y O U 41