Analytical Instrument Qualification According to USP <1058>: Requirements and Examples for the Agilent 1290 Infinity LC System

Analytical Instrument Qualification According to USP <1058>: Requirements and Examples for the Agilent 1290 Infinity LC System 1) USP <1058>: Scope, approach, requirements 2) Qualification Examples for LC 1290 3) Selected performance tests Dr. Edgar Naegele Application Scientist Agilent Technologies, Germany 1

Design Qualification Selected HPLC specifications for design qualification. Selected specifications of the Agilent 1290 infinity it autosampler 2

Design Qualification Selected criteria i for vendor assessment. All requirements of this custom vendor assessment template are fulfilled by Agilent Technologies! X X X X X X X X 3

Installation Qualification 1) Template and examples to document completeness of shipment for IQ. 3) Equipment documentation for IQ. yes yes 2) Verification of construction requirements for IQ. 4

Installation Qualification Testing for Installation Qualification Verification of correct system installation for IQ. For this purpose p Agilent uses a certified standard! 5

Operation qualification - an example procedure HPLC - System Pump Injector Oven Detector Computer Control& Evaluation Flow rate Inj. Vol. Temperature Signal precision accuracy accuracy and/or noise Specifications of the Agilent 1290 infinity LC system Flow rate precision Inj. Vol. accuracy Temperature accuracy Signal and/or noise 0.07% 07% RSD or <025RSD 0.25 +/- 08 C 0.8 <+/ +/- 3x10-6 AU 0.005 SD at Precision 0.2 5.0 ml/min +/- 0.05 C from 5 20 µl < 0.50 RSD from 2 5 µl < 0.70 RSD from 1 2 µl at 230/4 nm, slit width 4nm, TC 2 s ASTM 6

Operation qualification - an example procedure Computer Control & Evaluation Generated Automatically Header Signed and dated Summary Test/Limit/Result/Status Configuration Module/FW/Serial No. & Software Verification Log Book Test Reports Sequence/Log Method Chromatograms/Spectra/Reports Results/Sample Summary 7

Gradient test depending on the volume of the mixers (blue-35µl, µ, red-135µl, µ,g green-300µl) µ ) mau Gradient (stepwise): Time % A % B 600 000 0.00 100.00 000 0.00 3.00 100.00 0.00 3.01 90.00 10.00 500 6.00 90.00 10.00 6.01 52.00 48.00 400 9.00 52.00 48.00 9.01 48.00 52.00 12.00 48.00 52.00 300 12.01 10.00 90.00 15.00 10.00 90.00 15.01 0.00 100.00 200 18.00 0.00 100.00 18.01 80 48.00 800 52.00 100 21.00 48.00 52.00 21.01 52.00 48.00 Influence of the mixer 24.00 52.00 48.00 0 24.01 100.00 0.00 27.00 100.00 0.00 0 5 10 15 20 25 Gradient Testing: Mobile Phase A: Water Mobile Phase B: Water + 0,5% Aceton (v/v) min

1290 Infinity Binary Pump Degasser High resolution pump di drives Silicon Carbide piston Channel A Solvent selection valve Purge valve Jet Weaver Mixer Multi layer heat exchanger Channel B Next Generation Technology 1200 bar @ 2.0 ml/min 800 bar @ 5.0 ml/min Minimum dwell volume: 10 µl / 45 µl Minimum Mixing Noise Maximum RT repeatability Automatic Purge Automatic correction of von micro leaks 9

1290 Infinity binary Pump Minimum dead volume and mixing noise 1200 Series Solvent A 1200 Series Solvent B Passive Damper 0-300 µl Passive Mixer 400 µl Low Noise ~ 800 µl dead volume 1290 Inifinity Solvent A 1290 Inifinity Solvent B Active Damping: 10 µl Jet Weaver Mixer: 35 µl Firmware Hardware Agilent microfluidic, Multilayer Technologie + Pump Pump + = 10 µl / 45 µl DV: 80x 20x / smaller 20x kleiner Niedriges Lower Noise Noise < 50% 10

Example: Performance Qualification Documentation ti of PQ tests. t ChemStation Report with detailed statistical information Amount S.D. : 0.035 RSD: 0.13 11

Performance qualification an example 300 0.889 0.818 400 0.685 0.617 500 0.713 0.761 0.451 600 0.551 mau AU 200 100 0 0 0.2 0.4 0.6 0.8 1 RSD % RT (n=10) RSD % Area (n=10) 1 0.01600 0.401 2 0 00769 0.00769 0 393 0.393 3 0.00517 0.429 4 0.00575 0.470 5 0 00577 0.00577 0 398 0.398 6 0.00538 0.463 7 0.00585 0.437 8 0.00571 0.518 9 0.00797 0.555 Mean 0.00725 0.450 min Sample: Check Check-out out sample with 9 phenones 12 Peak Column: 2.1 x 50 mm, 1.8 µm Gradient: 5 to 95 % B in 0.5 min Flow rate: 1.2 ml/min Injection volume: 1µL

Analytical Instrument Qualification According to USP <1058>: Requirements and Examples for the Agilent 1290 Infinity LC System 1) USP <1058>: Scope, approach, requirements 2) Qualification Examples for LC 1290 3) Selected performance tests and performance data 13

Application depended Performance Data and System Suitability - Experimental conditions for the Agilent 1290 infinity LC system Configuration of the 1290-System G4220A 1290 Infinity Binary pump with integrated vacuum degasser and different solvent mixers G4226A 1290 Infinity Autosampler G1316C 1290 Infinity Column Compartment G4212A 1290 Infinity Diode Array Detector Software: Chemstation B.04.02 Reference samples A liquid formulation of metoclopramide hydrochloride (1 mg/ml) comprising 0.01% concentration of impurities was used for all tests. Setup for testing 1. Determination of pump performance depending on dwell volumes by gradient tests 2. Establishment of a chromatographic separation to achieve data for long time evaluations 3. Similar peak pattern and resolution according to selected column with respect to particle size and column dimension and adapted to gradient shape and flow 4. Precision of areas must be < 2 % RSD. 5. Precision of retention times must be < 0.5 % RSD. 6. Linearity should be given at least with R2>0.999 Sample setup for testing Sample Purpose Injections Blank solution Verify baseline stability and identify artifacts 3 Suitability sample Verify precision of areas and 10 retention times for reference solution Calibration Verify linearity 3 for each level Highest concentration ti Verify Carry over 3 of each sample and Blank solution 14 Confidentiality Label

Separation of Metoclopramide and its impurities on different columns. A B C D A) Agilent TC C18, B) Eclipse Plus C18, C) Eclipse Plus C18, D) Waters BEH C18, 250*4.6mm, 5µm 150*2.1mm, 3.5µm 150*2,1mm, 1.8µm 150*2.1mm, 1.7µm Flow rate 1.058ml/min 1.058ml/min 0.221ml/min 0.221ml/min Gradient 0-25min 5-57% B 0-15min 5-57% B 0-15min 5-57% B 0-15min 5-57% B Temperature 37 C 37 C 37 C 37 C Injection 8µl 2µl 1µl 1µl volume Detection DAD, Signal 275/4, Reference 400/60, standard cell Data rate 2 Hz 2 Hz 40 Hz 40 Hz Maximum 138 bar 145 bar 310 bar 435 bar pressure Mobile phase A : 0.25% w/w Ammonium acetate in water Mobile phase B : Acetonitrile

Determination of the precision of areas and retention times Impurities in Metoclopramide p Determination of the precision of areas and retention times Retention times Areas Linearity Mean RSD Mean RSD R 2 Impurity 1 6.927 0.030 3.765 1.36 0.9994 Impurity 2 7.386 0.025 2.170 1.49 0.9997 Metoclopramid 8.170 0.039 10190.182 0.58 0.9998 Impurity 3 8.714 0.035 1.707 1.48 0.9993 Impurity 4 9.395 0.013 1.496 1.58 0.9993 Impurity 5 10.018 0.039 1.023 1.55 0.9990 Impurity 6 10.551 0.039 1.971 1.46 0.9993 16

Agilent 1290 Infinity Autosampler Flow-through design Advantage of the flow-through design No sample loss Flexible injection volume with high precision from 0.1 to 20 µl No need to exchange the sample loop Compromise of the flow-through design higher dead volume of 85 µl For the 1290 Agilent infinity LC system 125 µl dead volume if loop is in main path 17

Detection of carry over. A) Injection of sample with highest concentration of metoclopramide. B) Injection of a blank solvent sample. C) Carry-over specification. A Metoclopramide mau 1.5 1.25 1 DAD1 A, Sig=257,4 Ref=360,100 (F:\CHLORHEX100\WATER1200000014.D) DAD1 A, Sig=257,4 Ref=360,100 (F:\CHLORHEX100\CHLOREX30020013.D) Chlorhexidine 0.75 B 0.5 0.25 0-0.25-0.5 0.0028% carry over 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 min Column: 2.1x50mm Eclipse Plus C18, 1.8µm Sample: Chlorhexidine (1200ng/µl) dissolved in Water+0.1%TFA Mobile Phases: water+0.05%tfa, Acetonitrile+0.045%TFA Isocratic: 64 % A + 33 % B. Flow rate: 0,5ml/min, Stop time: 2.5min Injection volume: 1µl, external needle wash, water + 0.1%TFA, 10sec Column temperature: 50 C DAD: 257/4nm, Ref 360/100nm, 20Hz

Speed optimized separation of Metoclopramide and its impurities Zorbax Eclipse Plus C18 150*2 1mm 18µm Zorbax Eclipse Plus C18, 150 2.1mm, 1.8µm Flow rate : 0.44ml/min Gradient : 10 minutes

1290 Infinity Diode Array Detector Optofluidic Waveguides Non-coated fiber (fused silica) High light transmission by Total-Internal Reflection (TIR) Principle Advantage High Sensitivity at Lowest Cell Volume No RI (Refractive Index) and Thermal Effects (Solvent Temperature) Data Rate up to160 Hz (Multi Wave Length + Spectra) One Flow Cell for all Applications Cartridge Design for easy Handling 20

Conclusion The new Agilent Infinity LC is designed to provide highest speed, resolution and sensitivity. A new power range allows you to operate with any particle type, any column dimensions, or any mobile and stationary phase. The Agilent 1290 Infinity HPLC system is can be qualified according to the USP <1058> chapter and will meet highest requirements for every LC application. A selection of tests that can be established to evaluate a LC system were shown: For instance gradient testing, the determination of precision of areas and retention and the determination of flinearity. it All results show explicitly the applicability of the 1290 Infinity LC system for quality control testing as well as in the FDA regulated environment. Agilent Services qualifies your system according to USP1058! 21

References/Further reading United States Pharmacopiea, Chapter <1058>, Analytical Instrument Qualification, USA, 2008 Analytical Instrument Qualification and System Validation, January 1, 2009, Agilent Publication Number 5990-3288EN Increasing productivity in the analysis of impurities iti in metoclopramide hydrochloride hl formulations using the Agilent 1290 Infinity LC system, Agilent Technologies Pub.-Number: 5990-3981EN Analytical instrument qualification and system validation according to USP Chapter <1058> for the Agilent 1290 Infinity LC system, Agilent Technologies Pub.-Number: 5990-4781EN 22

Thank you for your attention! Do you have Questions? 23