VALIDATION AND CONTROL OF BIOANALYTICAL MASS SPECTROMETRY METHODS

1. VALIDATION AND CONTROL OF BIOANALYTICAL MASS SPECTROMETRY METHODS

2. 02 ANTIDEPRESSANT DRUGS M.W.306.23 SERTRALINE NOR-SERTRALINE M.W.292.20

3. MS-MS SPECTRA of Sertraline and Nor-Sertraline Sertraline Nor-Sertraline

4. TIC (MRM) of Nor-Sertraline (A) and Sertraline (B)

5. Validation Categories • Pre-validation • Selectivity • Calibration • Detectability • Accuracy/Precision • Ruggedness/Stability

6. Validation Vs Quality Control Validation is the process of ensuring that a method is good enough for the intended purpose where as quality control ensures that a method is working during the analytical process.

7. SUMMARY OF CRYSTAL CITYCONFERENCE/DRAFT FDA GUIDANCE • Reference standards must be authenticated. • Each step in a procedure should be investigated for effect on results (from time of sample collection). • Stability should be assessed in each matrix under storage conditions (long and short term) and through 3 freeze thaw cycles. Stock solution and auto sampler stability should also be assessed. • Specificity with evidence of identification in six independent sources of each matrix under controlled conditions. No more than 10% may show significant (>20% LOQ?) interference.

8. SUMMARY CONT. • Concentration range must be defined by standards. • LOQ established with n=5 controls, independent of standards (acceptable accuracy and precision) • 15% (20% at LOQ) accuracy criteria (minimum n=5). • 15% (20% at LOQ) precision criteria. (minimum n=5) • A blank and 5-8 standards in singlicate ar replicate with each run of samples. • The simplest workable relationship should be used as a calibration model. • Unknown samples should be run in singlicate.

9. SUMMARY CONT. • Extrapolation of calibration not recommended. • The conformance of calibration data to the model should be tested (15%/20% residual, with 4 of 6 standards meeting the criteria). • System suitability should be addressed. • QC samples in duplicate at 3 concentrations (3 LOQ, midway, 75-90% of highest calib.). • 2/3 of controls (not the same concentration) must be within 20% of nominal value for acceptance of an analytical run. • Accuracy and precision of runs should be within ± 15% (20% at LOQ) for intra and inter-run data. • Reports should include 20% of unknown chromatograms.

10. PRACTICAL STATISTIC FOR ANALYTICAL CHEMISTS Figure 1.1 Good accuracy; good precision Figure 1.2 Poor accuracy; good precision Figure 1.3 Good accuracy; poor precision

11. Internal Standards Appropriate Assumptions for Use of Internal Standards 1.Qualitative marker 2. Monitor extractions (isotope) 3. Correct dilution errors 4. Monitor reactions (isotope) Inappropriate 1. Monitor extractions (Homologue) 2. Monitor detector Stability (must test) 3. Monitor reactivity (homologue)

12. Detectability

13. Detectability Definitions • Limit of Detection (LOD) - The lowest concentration that can be distinguished from zero with great probability. • Limit of ldentification (LOI) - The lowest concentration which can be determined to be present in a sample with great probability. • Limit of Quantitation (LOQ) - The lowest concentration that can be reliably quantitated. • Sensitivity - The slope of the analytical calibration curve.

14. Detectability Enhancement • Increase sample volume • Increase injected volume • Replicate Analysis (precision will improve by a factor of 1/n)

15. Desirable Characteristics of Internal Standards 1. Baseline resolution 2. Elution near peak of interest 3. Similar peak size 4. Chemically similar 5. Chemically stable 6. Should not be a metabolite ar degradation product 7. Similar "partition" behavior

16. Terms for Data Consistency • Precision - The doseness ofreplicate measures using the same method and conditions. • Reproductibility - The closeness of replicate measures using the same method under different conditions. • Repeatability - The closeness of agreement of successive measurements on the same sample.

17. Within Run Precision

18. Ruggedness

19. A-Priori Ruggedness Testing Column Effect ======> Compare results from 2 column tons. Mobile Phase Effects =======> Vary mobile phase components by ± 2%. Also Included ======> pH Temperature for GC Flow Acceptability =========> Acceptable peak shape and resolution with no loss of LLOQ. (Dadgar et al.1995)

20. Reasons for Cross Validation • New personnel • New matrix • New sample volume • Extended calibration range • Manual method changed to robotic • Time has elapsed (3 months) Solenger in Riley and Rosanske, Pergamon, 1996

21. Stability

22. Stability Studies • Sample collection • Sample transportation • Long-term storage • Short term in process • Freeze thaw • Anlytical process (autosampler) • Stock solution

23. STABILITY TEST GUIDELINES • Test conditions and materials should he completely described and duplicated. Standard materials should he primary standard grade with defined purity levels. The type of container temperature and presence or absence of light are important factors to document. • The method used for stability assessment must he detemined to he stability indicating. This means that decomposition products do not interfere with tire analysis of the intact compound. Tire stability indicating nature of the method can be demonstrated by analysis of known decomposition products or by subjecting the compound to severe stress as in intentionally decompose it. • Tire time zero determination must he rigorously established. It is not sufficient to rely on a prepared concentration value.

24. STABILITY TEST GUIDELINES • Multiple assays (e.g., 5-10) must be carried out to provide tire replicates needed for establishment of a reliable mean. • Tire entire concentration range in question should be covered because significantly different rates of decomposition and physical instability may occur at different concentrations. • Blank matrix samples should he run in conjunction with stability samples to ensure the absence of interferences. • Freshly prepared standard solutions, which are matrix matched and are preferably from tire some stock solution should he used to evaluate concentrations.

25. STABILITY ACCEPTANCE Ytest x 100or Test x 100 = % recovery x Yref ref ] Upper 90% Confidence < 90% Limit ] Lower 90% Confidence < 111% Limit

26. Recovery Studies 1. Direct - Extracted Unextracted X 100 = Rec. 2. Radioactive analog - Extracted counts Pré-extracted counts X 100 = Rec. 3. Spiked Residue - Extracted Unextracted Spiked residue X 100 = Rec. 4. Internal Standard (post-extraction)- Extracted ratio Unextracted radio X 100 = Rec.

27. Relative Recovery 1. Matrix vs Solvent Matrix extract Solvent extract X 100 = %Rec. 2. Accuracy Assayed conc. Prepared conc. X 100 = Rec. 3. Internal Standard (post-extraction)- Radio extracted Radtio unextracted X 100 = Rec.

28. SOP Number: 08/00Version Number: 1.0Current version issued at: 31/05/2000Written by: John Salmon, Jaime Ilha & Mauro SucupiraApproved by: Gilberto de Nucci STANDARDOPERATINGPROCEDURES The determination of Amoxicillin in Human Plasma by LC-MS-MS using Cefadroxil as the Internal Standard

29. Normal Plasma 0800Bt001 MRM of 2 Channels ES+ 2.52 366.00 > 349.20 2.45 2.29 3.11 1.85 2.19 2.88 100 1.92 2.93 3.25 1.66 2.75 2.33e3 % 0 0800Bt001 MRM of 2 Channels ES+ 2.24 2.38 3.14 3.21 1.78 1.92 2.58 2.74 364.00 > 207.80 2.93 100 1.64 2.10 2.68 2.17e3 % 0 0800Bt001 MRM of 2 Channels ES+ 1.85 1.92 2.38 2.45 2.88 3.41 2.24 2.93 3.46 TIC 1.64 3.14 3.21 3.11 2.58 100 2.10 2.61 3.99e3 % 0 Time 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40

30. Lipemic Plasma 0800Bt008 MRM of 2 Channels ES+ 2.45 3.09 366.00 > 349.20 1.88 3.34 100 1.58 2.84 2.24 2.77 2.87 1.79 2.29 3.14 2.17 1.62e3 2.52 2.63 % 0 0800Bt008 MRM of 2 Channels ES+ 1.78 3.32 364.00 > 207.80 3.16 1.64 100 2.68 2.43 2.57 3.12 2.84 1.90 2.04 2.34 2.91 2.20 1.85 3.46 1.23e3 % 0 0800Bt008 MRM of 2 Channels ES+ 1.78 2.84 3.03 3.09 TIC 2.52 3.39 1.64 2.33 2.45 3.32 100 1.88 2.29 2.04 2.70 2.07e3 % 0 Time 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40

31. Hemolysed Plasma 0800Bt004 MRM of 2 Channels ES+ 3.02 1.69 2.31 366.00 > 349.20 2.86 1.74 2.49 3.09 100 3.29 2.72 1.97 2.17 1.78 2.70e3 % 0 0800Bt004 MRM of 2 Channels ES+ 1.76 2.74 2.15 2.59 2.91 364.00 > 207.80 2.06 2.20 3.14 3.11 3.21 2.42 100 2.20e3 % 0 0800Bt004 MRM of 2 Channels ES+ 2.31 TIC 3.09 3.29 1.67 1.78 2.49 2.91 2.10 2.22 2.72 2.86 3.36 100 1.97 4.52e3 % 0 Time 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40

32. Analyte and Internal Standard QL1- 0.05ug/ml in plasma ext (spe) 0800v026 Sm (Mn, 2x5) MRM of 2 Channels ES+ 2.63 366.00 > 349.20 100 539 2.05e3 Area % 0 Time 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 QL1- 0.05ug/ml in plasma ext (spe) 0800v026 Sm (Mn, 2x5) MRM of 2 Channels ES+ 2.63 364.00 > 207.80 100 18946 8.05e4 Area % 0 Time 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

33. Table – LOQ validation data Validation List ID V01 Sample Code   QL1 Sample # Quantified Individual Concentration Precision 25 0.042 µg/mL 84.0% 26 0.044 µg/mL 88.0% 27 0.051 µg/mL 102.0% 28 0.062 µg/mL 124.0% 29 0.039 µg/mL 78.0% 30 0.042 µg/mL 84.0% 31 0.047 µg/mL 94.0% 32 0.047 µg/mL 94.0% Mean (µg/mL)0.04675 Precision (CV%)15.4% Accuracy (%)93.5%

34. Table – Calibration Curve Validation Data

35. Table - Quality Control Samples Definition

36. Table – Intra-batch Validation Data

37. Introduction This document is composed of two main parts as follows: • CHAPTER I- Assay Method – Description of the methodology developed by the Cartesius Analytical Unit used for the determination of Amoxicillin in Human Plasma by LC-MS-MS using Cefadroxil as Internal Standard • CHAPTER II- Method Validation Protocol & Results – Data related to the validation of the assay method, as follows: A description of the routine followed by the Cartesius Analytical Unit, to develop and validate the assay method; Specific results certifying the compliance of the method to those standards.

38. CHAPTER I 1 PRINCIPLES OF THE METHOD ................................ ......................………......... 1 SAFETY ................................ ................................ .......................……………........ 1 2 MATERIALS ................................ ................................ ..........................………...... 1 3 3.1 Apparatus ................................ ................................ .............................………………. 1 3.2 Standards ................................ ................................ ..........................……………….... 3 3.3 Reagents ................................ ................................ ................................ ................................ 3 3.4 Biological Specimens ......................... ................................ .............................…….. 3 ................................ ........……….................... 3 PREPARATION OF SOLUTIONS 4 4.1 Master Standard Solutions ................................ ................................ ................................ 3 4.1.1 Amoxicillin Master Standard Sol ution ................................ ...........……………................... 4 4.1.2 Cefadroxil Master Standard Solution ...................... ..........................……………...... 4 4.2 Working Solutions ................................ ................................ ........................………...... 4 4.2.1 Amoxicillin Working Solutions ................................ ................................ .................…-............ 5 4.2.2 Cefadroxil Worki ng Solutions ................................ ................................ ............…................... 5

39. CHAPTER I 4.3 Other Solutions ................................ ................................ ..........................………………..... 5 4.3.1 5 Mobile Phase & Elution Solution: 50% acetonitrile, 50% water & 10mM formic acid 5 PREPARATION OF CALIBRATION STANDARDS & QUALITY CONTROL SAMPLES ................................ ................................ ................................ ................................ 6 5.1 Preparation of Calibration Standards ................................ .......................……………....... 6 5.2 Preparation of Quality Control Samples ................................ .........................…………...... 7 6 PREPARATION OF ANALYTICAL RUNS .....................………….......... 8 6.1 Sample List Construction ....................... ……………………................................. 8 6.2 Sample Extraction ....................……………………………………………............ ................................ ................................ 9 6.3 Chromatographic Conditions ..................… ......................………………….......... 10 6.4 Mass Spectrometric Conditions ................................ ..........................………………….... 11 7 PRE-STUDY VALIDATION ................................ ........................………………........ 11

40. CHAPTER I 8 CONDUCTION, EVALUATION AND INTRA-BATCH VALIDATION OF ANALYTICAL RUNS ................................ ................................ ................................ ........…........ 12 8.1 Calculation ................................ ................................ ................................ ................................ .. 12 8.2 Validation of an Analytical Run ................................ ......................…………….......... 12 8.2.1 Initial Concerns ................................ .....................…………………………………........... 12 8.2.2 Calibration Curve ................................ ................................ ................................ ..............…................. 13 8.2.3 Quality Controls ................................ ................................ ................................ ........................……...... 14 8.2.4 Samples ................................ ................................ ................................ ................................ ................ 14 8.3 Rejection of individual test samples ................................ ..........................……………... 14 8.3.1 Data Rejection ................................ ................................ ................................ ................................ 14 8.3.2 Reassays ................................ ................................ ................................ ................................ ............... 15 8.3.3 Missing points ................................ ................................ ................................ ................................ 15

41. CHAPTER I 8.4 Determination of the estimated sample concentration ................................ ......................... 15 8.4.1 Multiple analysis ................................ ................................ ................................ ................................ .. 16 8.4.2 Single analysis ................................ ................................ ................................ ................................ ..... 16 8.4.3 Values at the end of the curve and below LOQ ................................ ................................ ................. 16 8.4.4 Values above the highest standard ................................ ................................ ................................ ...... 16 9 BETWEEN-BATCH STUDY VALIDATION ................................ ................................ ... 17 10 STABILITY CONSIDERATIONS ................................ ................................ ................. 17 10.1 Autosampler Stability ................................ ................................ ................................ ................ 17 10.2 Freeze and Thaw Stability ................................ ................................ ................................ ........ 17 10.3 Short Term Room Stability ................................ ................................ ................................ ...... 17 10.4 Long-Term Stability ................................ ................................ ................................ .................. 18 10.5 Stock Solution Stability ................................ ................................ ................................ ............. 18

42. CHAPTER II 1 PRE-STUDY VALIDATION ................................ ...........................…………... 19 1.1 Specificity ................................ ................................ ........................……………...... .... 20 1.2 Calibration Curve ................................ ................................ ............................……... 22 1.2.1 Determination of the Limit of Quantification ................................ .................................. 22 1.2.2 Linearity ................................ ................................ ................................ ................................ 24 1.3 Precision and Accuracy ................................ ................................ 26 1.3.1 Determination of the Quality Control Concentrations ..........................…………..... ....... 26 1.3.2 Intra-batch Validation ................................ ................................ ................................ .......................... 27 1.3.3 Inter-batch Validation ................................ ................................ ................................ .......................... 28 1.4 Recovery ................................ ................................ ..........................…………..... ...... 29

43. CHAPTER II 2 STABILITY ................................ ................................ ............................…….. 29 2.1 Autosampler Stability ................................ ................................ ................................ 30 2.2 Freeze and Thaw Stability ................................ ............................…………... 30 2.3 Short Term Room Temperature Stability ............................……………... 31 2.4 Long Term Stability ................................ ................................ ................................ 32 2.5 Stock Solution Stability ................................ ............................……………... 32 3 Conclusion ................................ ................................ ...........................…….... 33