Qualification of Analytical Equipments

it contains Qualification protocol of gas chromatography, HPLC, Infrared spectroscopy and LC-MS.

1. Qualification of Analytical Equipments Prepared by: Dhawal Rajdev M. Pharm (Q.A.) 2017-2019

2. Qualification of Analytical Equipments Gas chromatography 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: 1. Design qualification(DQ). 2. Installation qualification(IQ). 3. Operational qualification(OQ). 4. Performance qualification(PQ). 3

4. 1.Design qualification(D.Q): 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. 2.Installation Qualification (I.Q):  The purpose of I.Q is to check the installation site/ environment, confirms equipment specifications and verifies the condition of installed equipment. 3.Operational Qualification (O.Q):  O.Q includes procedures and documentation of O.Q of analytical instrument.  When all procedures are executed and all items pass the inspection, it is verified that the system operates to satisfy the intended purpose. 4

5. 4.Performance Qualification (P.Q):  The objective is to ensure that the instrument is performing within specified limits.  Hence documented verification that the equipment and ancillary systems, as connected together, can perform effectively and reproducibly based on the approved process method and specifications.

6. Qualification of Gas Chromatography Level I. Selection Of Instruments & Suppliers At level I of the qualification of a gc equipment(selection of instruments and suppliers) It is recommended to select a manufacture of gc that can satisfy the needs of the laboratory and works under ISO 9001 certification Level II of Equipment Qualification : 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 by appropriately qualified personnel, using certified reference buffers according to an approved procedure. 6

7. Level III. Periodic and motivated instrument Checks Examples of requirements for GC instruments with FID 7 instrument module Parameter to be checked Typical tolerance limit 1. Inlet system 1.1 Injector leak test 1.2. Pressure/flow accuracy and stability 1.3. Repeatability of injection (overall test 1) - In split mode -In split less mode 1.4. Injector temperature accuracy and stability 1.5. Carry-over (overall test 3) Pressure drop ≤ 15 kPa within 5 minutes Covered by overall test 1 RSD ≤ 3.0% RSD ≤ 3.0% Covered by overall test 2 ≤ 0.2

8. instrument module Parameter to be checked Typical tolerance limit 2. Oven 2.1. Repeatability of oven temperature characteristics Covered by overall test 2 3. FID detector 3.1. Linearity (overall test 3) 3.2. Constant detector response 3.3. Noise 3.3. Drift r 2 ≥ 0.999 Covered by overall test 1 or 2 8

9. Level III. Periodic and motivated instrument checks Practical examples of tests and their associated tolerance limits for several parameters related to the performance of the different modules of a GC are presented below. These examples can be considered by the OMCLs as possible approaches to perform the Level III of the equipment qualification process: “Periodic and motivated instrument checks”. Several tests are proposed to check various parameters at the same time (overall tests). In order to run the tests in a more economical way, other suitable solutions can be used, as for example, the “ Grob Test” mixture, available from different suppliers (e.g. Alltech , Sigma, Thames Restek ). This commercial solution should be appropriate to the column material used. It is recommended to run the overall tests by using always the same test column, exclusively dedicated to qualification purposes, to guarantee reproducible conditions 9

10. 1 . Inlet system The following tests are proposed for the periodic and motivated check of the GC Inlet System. 1.1. Injector leak test Method:  If not otherwise specified by the instrument manufacturer, the leak test is carried out according to the procedure laid down in the instrument manual or by the built in automatic leak check procedure of the instrument.  Otherwise use the test described below:  Disconnect the column from the injector and close the injector outlet with a sealed cap.  Close the septum purge and the bypass.  Adjust the flow and pressure controller to the maximal possible value of the pressure gauge. 10

11.  Adjust the flow controller to zero.  Read the pressure after 1 minute and record the value.  Record the pressure after 5 minutes. Limits:  Pressure drop ≤ 15 kPa within 5 minutes. 1.2. Inlet pressure/flow accuracy and stability  A direct measurement of these parameters was not deemed practical or necessary, but the optimal conditions of flow/pressure can be verified by the overall test 1. Limits: Refer to overall test 1 1.3. Repeatability of injection  The verification of this parameter is covered by the overall test 1.  This test is to be performed in both split and split less mode. Limits: Refer to overall test 1.

12. 1.4. Injector temperature accuracy and stability  Due to the fact that the temperature cannot be reliably measured without opening and modifying the system and due to the difficulties of introducing a probe inside this module, the verification of this parameter is considered to be covered by the overall test 2. Limits: Refer to overall test 2. 1.5. Injector carry over  After having injected the solutions for the linearity test of the FID detector, in increasing order, inject the blank and measure the peaks that correspond to the major peaks (= analytes) in the linearity solutions.  The verification of this parameter is covered by the overall test 3. Limits: Refer to overall test 3 12

13. 2. OVEN 2.1. Repeatability of the oven temperature characteristics  Due to the fact that the temperature cannot be reliably measured without opening and modifying the system conditions and that even when introducing a probe inside the oven, its location would not reflect the real temperature conditions at all points, the verification of this parameter is covered by the overall tests 2A and 2B. Limits: Refer to overall test 2. 3. FID DETECTOR The following tests are proposed for the periodic and motivated check of the GC FID detector. 3.1. FID detector linearity  Increasing amounts of analyte are injected and a linear response should be obtained.  The verification of this parameter is covered by the overall test 3. Limits: Refer to overall test 3. 13

14. 3.2. Constant FID detector response  The proper and reproducible functioning of the FID can be demonstrated by checking the peak areas obtained from a predefined standard solution.  The verification of this parameter is covered by the overall test 1 or 2. Limits: Refer to overall test 1 or 2. 3.3. Fid detector noise and drift  If the instrument has a built-in automatic system for the verification of the noise and drift, follow the manufacturer’s instructions and apply the defined acceptance criteria. Otherwise, use the test described below: Settings:  Column installed  Suitable flow, depending on column length/diameter  No injection  Oven temperature: 40°C  Detector on and heated at working temperature (270- 300°C)

15. 3.3. Fid detector noise and drift  If the instrument has a built-in automatic system for the verification of the noise and drift, follow the manufacturer’s instructions and apply the defined acceptance criteria. Otherwise, use the test described below: Settings:  Column installed  Suitable flow, depending on column length/diameter  No injection  Oven temperature: 40°C  Detector on and heated at working temperature (270- 300°C) 15

16. Method:  After stabilisation of the system, record the signal for 15 minutes.  Noise: evaluate 10 periods of 1 minute and calculate the mean value.  Drift: Evaluate the slope of the baseline over the 15 minutes. Limits:  The acceptance criteria for these parameters have to be chosen in accordance with the instrument vendor’s instructions and the intended use of the instrument.  If no instructions are given, the user has to pre-define these acceptance criteria by taking into account the previous experience and the intended use of the instrument.  No fixed values can be pre-defined in this guideline due to the high variety of integration systems used and consequently the acceptance criteria may be expressed in different units (voltage, current, arbitrary units per time).

17. OVERALL TEST 1  The overall test 1 covers the following parameters: - Pressure/flow accuracy and stability in the inlet system: Retention time repeatability - Repeatability of injection: peak area precision - In split mode - In split less mode The test may be combined with overall test 3. Split mode :  Test solution: 1-octanol in n-hexane 1% (v/v) Settings:  Column: SPB-1 (30m x 0.32mm ID x 0.25µm film)  Carrier gas: He  Velocity: 25cm/sec  Split: 1:100  Injection: 1µl 17

18. Injector temperature: 220°C  Oven temperature: 100°C isotherm  Detector temperature: 300°C  Runtime: 8 min  Retention time of 1-octanol: about 5 min Split less mode:  Stock solution: 1-octanol in n-hexane 1% (v/v)  Test solution: Dilute 10 ml of the stock solution with nhexane to 100 ml (corresponds to 1µl/ml of 1-octanol in nhexane ) Settings:  Column: SPB-1, 30m, 0.32mm ID, 0.25µm film  Carrier: He  Velocity: 30cm/sec  Split less injection: purge valve closed during 2 min  Injection: 0.2µl of the test solution 

19. Injector Temperature: 220°C  Oven Temperature: Initial 60°C for 4 min, 15°C/min. up to 135°C, final time 1min  Detector temperature: 300°C  Runtime: 9.5 min  Retention time of 1-octanol: about 8 min Method: Carry out 6 consecutive injections of the test solution and calculate the RSD of the different peak areas and retention times. Limits:  Retention time repeatability: the RSD of the retention times should be ≤ 2.0%  Peak area precision (split and split less mode): the RSD of the peak areas should be ≤ 3.0%

20. OVERALL TEST 2  The overall test 2 covers the following parameters: - Injector, oven and detector temperature accuracy and stability: retention time repeatability - Two alternative tests are proposed 1.Overall test 2A Test solution:  0.035 ml 1-octanol  0.035 ml 2-octanone  0.035 ml 2,6-dimethylanilin  0.035 ml n- tridecane  0.035 ml n-tetradecane  35 mg n- eicosane Dissolved in 50 ml Dichloromethane

21. Settings:  Column: SPB-1 (30m x 0.32mm ID x 0.25µm film)  Carrier gas: Helium  Velocity: 25 cm/s  Split: 1:100  Injection volume: 1 µl  Injector temperature: 220°C  Detector: FID  Detector temperature: 300°C  Gradient programme : 60°C (4 min), 5°C/min, 270°C (3 min) Method:  Inject the solution twice and calculate the relative retention times in relation to n- eicosane (RRT = 1)  The above table shows the approximately expected relative retention times. 21

22. Method:  Inject the solution twice and calculate the relative retention times in relation to n- eicosane (RRT = 1)  The above table shows the approximately expected relative retention times . Limits: The RSD of each RRT from two consecutive injections should be ≤ 1.0% 22 Analyte 1-octanol 2-octanone N- tridecane N-tetra decan RRT 0.30 0.22 0.52 0.60

23. 2.Overall test 2B Test Solution: 1.0% (W/W) n- Nonane and Hexadecane in Tetradecane. Settings:  Column: Ultra-1 (25m x 0.32mm ID x 0.52µm film)  Injection volume: 1 µl  Solvent: Tetradecane  Oven temperature: 110°C  Gradient programme : 110°C, 20°C/min, 180°C (final time: 3.5 min) Detector temperature: 250°C  Injector temperature: 200°C  Detector: FID

24. Flow rates: Carrier gas (Helium): 2 ± 0.2 ml/min Hydrogen: 30 ± 1.0 ml/min  Air: 400 ± 20.0 ml/min  Makeup (Nitrogen): 28 ± 1.0 ml/min  Split ratio: 15  Split vent: 30 ± 3.0 ml/min  Septum purge: 3-5 ml/min Method:  Allow the system to equilibrate  Injection sequence: 1) Blank (Tetradecane) 2) 6 replicates of the test solution. 24

25. Calculate the mean of the retention times and peak areas and the relative standard deviation of n- Nonane and n-Hexadecane Limits:  Retention time repeatability: RSD of the peak retention times of the 6 replicates ≤ 2%  Retention time ( Rt ) accuracy: for this example, the retention time ranges shown in the table below are proposed. Nevertheless, individual ranges should be predefined by the laboratory depending on the column used (e.g. Rt ± 0.2 min). OVERALL TEST 3  This test is a modified version of the overall test 1 to be used for the verification of: - Detector linearity: linearity of the areas recorded - Injector carry-over: area recorded in the blank run  It is described for both split and split less mode and may be combined with overall test 1.

26. Split mode :  Test solution: 1-octanol in n-hexane 1% (v/v)  Prepare further reference solutions by diluting the test solution as described below. Settings: see overall test 1  Injection sequence:  5.0 ml of the test solution diluted to 25.0 ml with n-hexane (2 µl/ml): 2 injections 10.0 ml of the test solution diluted to 25.0 ml with nhexane (4 µl/ml): 2 injections  15.0 ml of the test solution diluted to 25.0 ml with n-hexane (6 µl/ml): 2 injections  20.0 ml of the test solution diluted to 25.0 ml with n-hexane (8 µl/ml): 2 injections  If combined with overall test 1 for repeatability: test solution (10 µl/ml): 6 injections n-hexane as blank (carry over) 26

27. Split less mode:  Stock solution: 1-octanol in n-hexane 1% (v/v)  Test solution: Dilute 10 ml of the stock solution with nhexane to 100 ml (corresponds to 1µl/ml of 1-octanol in nhexane ).  Prepare further reference solutions by diluting the test solution with n-hexane.  Settings: see overall test 1 Injection sequence:  5.0 ml of the test solution diluted to 25.0 ml with n-hexane (0.2 µl/ml): 2 injections  10.0 ml of the test solution diluted to 25.0 ml with n-hexane (0.4 µl/ml): 2 injections  15.0 ml of the test solution diluted to 25.0 ml with n-hexane (0.6 µl/ml): 2 injections  20.0 ml of the test solution diluted to 25.0 ml with n-hexane (0.8 µl/ml): 2 injections  If combined with overall test 1 for repeatability: test solution (1 µl/ml): 6 injections n-hexane as blank (carry over)

28.  Linearity: coefficient of correlation of the calibration line obtained with the reference solutions and the test solution: r 2 ≥ 0.999. Carry-over: the percentage of the peak area corresponding to the analyte in the blank solution should be ≤ 0.2% of the peak area of this analyte in the chromatogram obtained with the solution with the highest concentration within the sequence 28

29. Level IV. In-use instrument checks Examples of requirements for GC instruments with FID 29 Parameter to be checked Typical tolerance limit 1. System suitability check for the method According to Ph. Eur. or MAH dossier or validated in-house method 2. Peak area precision RSD ≤ 3.0% unless otherwise prescribed* 3. Retention time repeatability RSD ≤ 2.0% 4. Sensitivity (where relevant, e.g. for related substances tests) According to Ph. Eur. or MAH dossier or validated in-house method

30. Reference:  Ph.Eur.2.2.35 chromatography; gas chromatography  Guidance on equipment qualification of analytical instruments  Journal of Perkin Elmer life & analytical science

31. Qualification of Analytical Equipments High Performance Liquid Chromatography 31

32. 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: 1. Design qualification(DQ). 2. Installation qualification(IQ). 3. Operational qualification(OQ). 4. Performance qualification(PQ). 32

33. 1.Design qualification(D.Q): 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. 2.Installation Qualification (I.Q):  The purpose of I.Q is to check the installation site/ environment, confirms equipment specifications and verifies the condition of installed equipment. 3.Operational Qualification (O.Q):  O.Q includes procedures and documentation of O.Q of analytical instrument.  When all procedures are executed and all items pass the inspection, it is verified that the system operates to satisfy the intended purpose. 33

34. 4.Performance Qualification (P.Q):  The objective is to ensure that the instrument is performing within specified limits.  Hence documented verification that the equipment and ancillary systems, as connected together, can perform effectively and reproducibly based on the approved process method and specifications.

35. Qualification of HPLC 1. Design qualification: 35 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.

36. Design element Examples 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 4 36

37. Design element Examples Operational specification •Detector: base line noise:<5 x 10-5 AU •Sampler: precision inj. Volume : <0.5% RSD. Sample carry over:<0.5% •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 37

38. 2.INSTALLATION QUALIFICATION : Installation qualification establishes that the instrument is received as design and specified. •It establishes that instrument is properly installed in the selected environment and that environment should be suitable for operation of the instrument. •Run of test samples verifies correct installation of all modules, electrical and fluid connections BEFORE INSTALLATION ? Obtain manufacturers recommendations for installation site requirements Check the site for the fulfillment of the manufacturers recommendation (utilities such as electricity and environment condition such as humidity and temperature) Allow sufficient shelf space for the equipment, SOPs, operating manual and software. 38

39. 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 tubings , for fluid connections , column in HPLC , power cables , data flow and instrument control table) 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.

40. 3.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. Eg : 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. 40

41. Test parameters and acceptance criteria : 41 Parameter Procedure User limit Leak testing Flow test by volume or weight/time ±5% Baseline drift ASTM (American society for testing material) method E19.09, 20 min <2 x 10-3 AU Baseline noise ASTM method E19.09, 20 min x 1 <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

42. PARAMETER PROCEDURE USER LIMIT Detector linearity Inject 5 standards >1.5 AU, 5% RSD Wavelength accuracy Holmium oxide filter ±1 nm Temperature accuracy Comparison with external 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% with acetone tracer ±1% 42

43. 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. 43

44. 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 103 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. 44

45. 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.

46. 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. 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 46

47. 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 Temperature precision: Monitor temperature for 20minutes and limit should be in between ±0.25 ºc

48. 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%. All compositions accuracies are calculated as the absolute difference between the mean composition at each set point and the theoretical composition. 48

49. 4.Performance qualification : It is the process of demonstrating that an instrument consistently performs according to a specification appropriate for its routine use. Important here is the word consistently. The test frequency is much higher than for OQ. Another difference is that PQ should always be performed under conditions that are similar to routine sample analysis. For a chromatogram this means using the same column, the same analysis conditions and the same or similar test compounds. PQ should be performed on a daily basis or whenever the instrument is used. The test frequency of only depends on the stability of the equipment but on everything in the system that may contribute to analysis the result For a liquid chromatograph, this may be the chromatographic column or a detectors lamp 49

50. 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 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.

51. Test should include: •Precision of the amounts •Precision of retention times •Resolution between two peaks •Peak width at half height •Peak tailing •Baseline noise •Wavelength accuracy of the uv /vis wavelength detector, preferably using built-in holmium – oxide filters REFERENCES: •Ph.Eur.2.2.35chromatography; High performance liquid chromatography •Guidance on equipment qualification of analytical instruments •Journal of Perkin Elmer life & analytical science 51

52. Qualification of Analytical Equipments Infra Rad Spectrophotometer 52

53. 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: 1. Design qualification(DQ). 2. Installation qualification(IQ). 3. Operational qualification(OQ). 4. Performance qualification(PQ). 53

54. 1.Design qualification(D.Q): 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. 2.Installation Qualification (I.Q):  The purpose of I.Q is to check the installation site/ environment, confirms equipment specifications and verifies the condition of installed equipment. 3.Operational Qualification (O.Q):  O.Q includes procedures and documentation of O.Q of analytical instrument.  When all procedures are executed and all items pass the inspection, it is verified that the system operates to satisfy the intended purpose. 54

55. 4.Performance Qualification (P.Q):  The objective is to ensure that the instrument is performing within specified limits.  Hence documented verification that the equipment and ancillary systems, as connected together, can perform effectively and reproducibly based on the approved process method and specifications. 55

56. QUALIFICATION OF IR SPECTROPHOTOMETER INTRODUCTION  The present document explains about “Qualification of Equipment(IR Spectrophotometer)”,  It should be used in combination with it when planning, performing and documenting the IR spectrophotometer qualification process.  The document contains the Introduction and general forms for Level I and II of qualification, which are common to all type of instruments.  For FTIR spectrometers, an example has been added to give instrument-specific proposals that may be used in combination with the general requirements presented in the core document “Qualification of Equipment”, when drawing up a Level I checklist. 56

57.  The present annex contains instrument-related recommendations on parameters to be checked at Level III and IV of qualification and the corresponding typical acceptance limits, as well as practical examples on the methodology that can be used to carry out these checks. Level I. Selection Of Instruments & Suppliers Example of check-list (Non-Exhaustive) Manufacturer:____________ Provider/ Distributor:___________ Name Of Instrument and Type: __________ 57

58. 58 Attribute (This list may be adapted if necessary) Specifications Benefits (Instrument/ supplier) Assessment (Pass/Fail) SPECTROPHOTO METER Detector range The optical bench shall include a DTGS detector with a frequency range of 7400 to 350 cm-1 It shall include a compressed air interferometer Spectral resolution The instrument shall come with an air-cooled standard infrared source.

59. Attribute (This list may be adapted if necessary) Specifications Benefits (Instrument/ supplier) Assessment (Pass/Fail) Wave number accuracy The instrument shall have a spectral resolution not exceeding 1.0 cm-1 The interferometer shall have at least four basic velocity levels: software shall permit the selection of a greater number of velocities between the basic levels. Wave number accuracy shall be better than +- 0.01 cm-1 The mirror’s greatest velocity shall allow a speed of at least five sweeps per second The laser and infrared beams must be coaxial to enable rapid, easy alignment of the system, depending on the samples or accessories 59

60. ughjhjkjk Attribute (This list may be adapted if necessary) Specifications Benefits (Instrument/ supplier) Assessment (Pass/Fail) The optical bench shall have main experimentation module with a device to allow purging with nitrogen At purchase, the main experimentation module shall be designed so it can receive 13 and 5 mm potassium bromide pellets 60 Notes: -  This check-list, containing examples of technical attributes that can be taken into account in the selection of an instrument and supplier, can be used in combination with the general check-list presented in Level I in the core document “Qualification of Equipment”.

61. Level II of Equipment Qualification: 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 by appropriately qualified personnel, using certified reference buffers according to an approved procedure. 61

62. Level III. Periodic & Motivated Instrument Checks Examples of requirements for IR spectrophotometers P arameters to be checked 1. Wave-number scale 2. Detector energy ratio 3. Signal/Noise ratio 4. Resolution 5. Zero test 6. Contamination check (only for ATR instruments) 7. Throughput check (only for ATR instruments) 62

63. 1.WAVE-NUMBER SCALE Method and Limits:  The wave-number scale may be verified by recording the spectrum of a polystyrene film, which has transmission minima (absorption maxima) at the wave numbers (in cm-1 ) shown in the table below: 63 TRANSMISSION MINIMA (cm -1) ACCEPTABLE TOLERANCE (cm -1) monochromatic instruments FTI±R instruments 3060.0 ±1.5 ±1.0 2849.5 ±2.0 ±1.0 1942.9 ±1.0 ±1.0 1601.2 ±1.0 ±1.0 1583.0 ±1.0 ±1.0 1154.5 ±1.0 ±1.0 1028.3 ±1.0 ±1.0

64. 2. DETECTOR ENERGY RATIO: Method:  Record the minimum energy ratio value for at least one of the following measurement points and compare it to the vendor’s specifications: - ◦ Energy at 3990 cm-1 / energy at 2000 cm-1 ◦ Energy at 4000 cm-1 / energy at 2000 cm-1 ◦ Energy at 3400 cm-1 / energy at 1300 cm-1 ◦ Energy at 2000 cm-1 / energy at 1000 cm-1 Limits:  Energy ratio test specifications vary for each spectrometer configuration.  The optical bench shall include a DTGS detector with a frequency range of 7400 to 350 cm-1 64

65. 3. SIGNAL/NOISE RATIO : Method:  Record the maximum noise level for each of the following regions: Peak-to-peak noise between: ◦ 4050 cm-1 and 3950 cm-1 ◦ 2050 cm-1 and 1950 cm-1 ◦ 1050 cm-1 and 950 cm-1 ◦ 550 cm-1 and 450 cm-1 (systems with DTGS detector only) RMS (root mean square) noise between: ◦ 4050 cm-1 and 3950 cm-1 ◦ 2050 cm-1 and 1950 cm-1 ◦ 1050 cm-1 and 950 cm-1 ◦ 550 cm-1 and 450 cm-1 (systems with DTGS detector only) Limits (% T):  Noise level test specifications vary for each spectrometer configuration. 65

66. 4. RESOLUTION :  Materials:  Certified polystyrene film of approximately 35 µm in thickness.  Method: - For instruments having a monochromator, record the spectrum of the polystyrene film. - For Fourier-transform instruments, use suitable instrument resolution with the appropriate apodisation prescribed by the manufacturer. The resolution is checked by suitable means, for example by recording the spectrum of a polystyrene film approximately 35 µm in thickness. Limits: -Difference between the absorbance's at the absorption minimum at 2870 cm-1 and the absorption maximum at 2849.5 cm-1 > 0.33. - Difference between the absorbance's at the absorption minimum at 1589 cm-1 and the absorption maximum at 1583 cm-1 > 0.08. 66

67. 5. ZERO TEST Method:  When using a polystyrene film of approximately 35 µm in thickness as standard at the wavelength of 2925 cm-1 and 700 cm-1 , almost complete absorption of the irradiated energy can be observed.  With this test, the remaining transmission is measured. As the maximum absorption can be observed at 700 cm-1 negative values may be observed.  The objective of the test is to evaluate if, despite the fact that there is almost complete absorption, energy is still detectable.  Non-valid results are an indication of a non-linear behaviour of the detector and the electronic system. 6. CONTAMINATION TEST (Only for Attenuated Total Reflection (ATR) instruments) Note: If an automated system is available, this test can be run more frequently or it can be transferred to Level IV, to be run before each analysis. 67

68. This test checks the presence of peaks that signal a contamination problem.  Use the automated function of the instrument (if available)to perform this test. If not available, record a background spectrum. Limits: Wave No.(cm-1) Upper limit(A) 3100-2800 0.1 1800-1600 0.1 1400-1100 0.2 68

69. 7. THROUGHPUT CHECK : (Only for Attenuated Total Reflection (ATR) instruments)  Note: If an automated system is available, this test can be run more frequently or it can be transferred to Level IV, to be run before each analysis. Method:  This test checks for an unexpected reduction of the transmittance.  An instrument specific automated test can be used.  A background spectrum is recorded and the transmittance is measured at 3 wave numbers e.g. 4000, 2600 and 1000 cm-1 . Limits:  The lower limit of the transmittance for the 3 wave numbers must be 80 % REFERENCES:  Ph Eur. 2.2.24, Absorption spectrophotometer, Infrared.  Guidance on equipment qualification of analytical instruments.  Journal of perkinelmer life and analytical sciences. 69

70. Qualification of Analytical Equipment Liquid Chromatography with Mass Spectrometer (LC-MS)

71. 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: 1. Design qualification(DQ). 2. Installation qualification(IQ). 3. Operational qualification(OQ). 4. Performance qualification(PQ). 71

72. 1.Design qualification(D.Q): 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. 2.Installation Qualification (I.Q):  The purpose of I.Q is to check the installation site/ environment, confirms equipment specifications and verifies the condition of installed equipment. 3.Operational Qualification (O.Q):  O.Q includes procedures and documentation of O.Q of analytical instrument.  When all procedures are executed and all items pass the inspection, it is verified that the system operates to satisfy the intended purpose. 72

73. 4.Performance Qualification (P.Q):  The objective is to ensure that the instrument is performing within specified limits.  Hence documented verification that the equipment and ancillary systems, as connected together, can perform effectively and reproducibly based on the approved process method and specifications. 73

74. Qualification of Mass Spectroscopy The context of validation for mass spectrometry (MS)-based methods is critically analysed . The focus is on the fitness for purpose depending on the task of the method. Information is given on commonly accepted procedures for the implementation and acceptance of analytical methods as 'confirmatory methods' according to EU criteria, and strategies for measurement. Beneficial aspects of the qualification process to ensure the suitability of the MS analytical system are also evaluated and discussed.

75. LEVEL I: Design Qualification: design qualification is is the documented collection of activities that define the functional and operational specification of the instrument and criteria for selection of vendor, based on intended performance of the instrument. The user should ensure that commercial off the shelf(COTS) instruments are suitable for their intended to application and that the manufacturer should adopt the quality systemthat provide for reliable equipment. All hardware and software laboratory products including operating system and software used to deliver qualification service are design, manufacturing and tested according to company's life cycle development procedure.

76. Level II: Operational Qualification: Temperature accuracy and stability of column heater/cooler Holmium oxide wavelength scan (if applicable) Detector lamp intensity and wavelength accuracy Detector noise and drift Pump flow rate accuracy and repeatability High and low pressure shutdown accuracy Injector precision Detector linearity and sample- to-sample carryover Injection volume linearity Gradient composition accuracy Linear gradient tested using IPA and 0.5% Acetone/IPA Five step gradient tested using IPA and 0.5% Acetone/IPA

77. Level III. Periodic and motivated instrument checks requirements for GC-EI-MS (examples) *) PFTBA (FC-43): Perfluoro-tributyl-amine (CAS NO.: 311-89-7) **) In case of quantification purposes, these parameters have to be checked Parameter to be checked Typical tolerance limits Mass accuracy PFTBA (FC-43) *) or internal calibration gas m/z = 69 ± 0.5 m/z = 219 ± 0.5 m/z = 502 ± 0.5 or defined masses of internal calibration gas ± 0.5 Linearity **) r² ≥ 0.995 System/instrument precision **) ≤ 10.0%

78. Level III contains practical examples of tests and their associated tolerance limits for several parameters related to the performance of GC-EI-MS. These examples can be considered by the OMCLs (Official Medicine Control Laboratory) as possible approaches to perform the Level III of the equipment qualification process: “Periodic and motivated instrument checks”. GENERAL CONSIDERATIONS - GC-MS is mainly used for the identification of unknown substances or quantification of low concentrated substances where high specificity is needed.

79. MASS ACCURACY Materials: PFTBA (FC-43) or internal calibration gas Method: Internal instrument check or spectrum of PFTBA (FC-43) in full scan mode Limits: m/z = 69 ± 0.5 m/z = 219 ± 0.5 m/z = 502 ± 0.5 Note : the tolerance limits for mass accuracy are only valid for quadrupole and ion trap mass spectrometers. Instruments like Time-of-Flight (TOF) or hybrid instruments like quadrupole Time-of-Flight (QTOF) have a much better mass accuracy and adequate requirements should be applied by the laboratory .

80. LINEARITY Materials: Stock solutions: 1-Octanol in dichloromethane 0.2, 0.4, 0.6, 0.8, 1.0 µL/mL Method: 2 injections of each level (injection volume: 1.0 µL) Limits: r² ≥ 0.995 SYSTEM/INSTRUMENT PRECISION Materials: Stock solution: 1-Octanol in dichloromethane 1.0 µL/mL Method: 6 injections (injection volume: 1.0 µL) Limits (minimum requirement): RSD ≤ 10.0% (without internal standard)

81. Level IV. In-use instrument checks Requirements for GC-EI-MS Parameter to be checked Typical tolerance limits As defined by the specific analysis method or according to the Ph. Eur. or the MAH dossier Identified by library or with reference standard

82. Level IV contains practical examples of tests and their associated tolerance limits for several parameters related to the performance of GC-EI-MS. These examples can be considered by the OMCLs as possible approaches to perform the Level IV of the equipment qualification process: “In-use instrument checks”. SYSTEM/INSTRUMENT PRECISION **) Materials: Any of the above-mentioned reference standard solutions used for the identification Method: 6 injections (injection volume: 1.0 µL) Limits (minimum requirement): RSD ≤ 10.0% **) in case of use for quantification Other tests according to the system suitability of the analysis method in use

83. Reference: https://shop.perkinelmer.com/Content/technicalinfo/tch_glpformatofhplcandlcmssystems.pdf OMCL Network of the Council of Europe QUALITY MANAGEMENT DOCUMENT PA/PH/OMCL (10) 86 2R QUALIFICATION OF EQUIPMENT ANNEX 7: QUALIFICATION OF MASS SPECTROMETERS Equipment Qualification Plan, Aligent Enterprice Edition Compliance Services.