The certification of the mass fraction of thyroxine in a CRM intended for calibration. Certified Reference Material IRMM-468

Transcription

1 The certification of the mass fraction of thyroxine in a CRM intended for calibration Certified Reference Material IRMM-468 B. Toussaint, C.L. Klein, M. Wiergowski Report EUR EN

2 The mission of IRMM is to promote a common and reliable European measurement system in support of EU policies. European Commission Directorate-General Joint Research Centre Institute for Reference Materials and Measurements Contact information European Commission Directorate-General Joint Research Centre Institute for Reference Materials and Measurements Retieseweg 111 B-2440 Geel Belgium Tel.: +32 (0) Fax: +32 (0) Legal Notice Neither the European Commission nor any person acting on behalf of the Commission is responsible for the use which might be made of the following information. A great deal of additional information on the European Union is available on the Internet. It can be accessed through the Europa server EUR Report EN Luxembourg: Office for Official Publications of the European Communities ISBN European Communities, 2006 Reproduction is authorised provided the source is acknowledged Printed in Belgium

3 European Commission IRMM information REFERENCE MATERIALS The certification of the mass fraction of thyroxine in a CRM intended for calibration Certified Reference Material IRMM-468 B. Toussaint, C.L. Klein, M. Wiergowski European Commission Directorate General Joint Research Centre Institute for Reference Materials and Measurements Reference Materials Unit Retieseweg 111 BE-2440 Geel, Belgium Report EUR EN

4 SUMMARY This report describes the preparation, homogeneity, short-term and long-term stability and characterisation studies of the certified reference material IRMM-468, consisting of thyroxine certified for its purity. A description of the analytical procedures used and all relevant data from the certification studies are presented. The certified mass fraction (expressed on dry mass basis) is: Substance Certified mass fraction 1) Uncertainty 2) thyroxine ) The certified value is the purity after taking into consideration inorganic residues, water, ethanol and organic impurities detectable by HPLC-UV and HPLC-MS. The certified value is traceable to the International System of Units (SI). 2) The certified uncertainty is the expanded uncertainty estimated in accordance with the Guide to the Expression of Uncertainty in Measurement (GUM) [1]. It is expressed with a coverage factor k = 2, corresponding to a level of confidence of about 95%. 1

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6 TABLE OF CONTENTS SUMMARY... 1 TABLE OF CONTENTS... 3 GLOSSARY INTRODUCTION BACKGROUND: NEED FOR THE CRM CHOICE OF THE MATERIAL TO BE CERTIFIED DESIGN OF THE PROJECT PARTICIPANTS STABILITY AND HOMOGENEITY STUDIES CHARACTERISATION MEASUREMENTS PREPARATION OF THE MATERIAL BOTTLING DISPATCHING OF SAMPLES TESTING OF THE MATERIAL HOMOGENEITY STUDY STABILITY STUDY Short-term stability study Long-term stability study CHARACTERISATION MEASUREMENTS CALIBRANTS ANALYTICAL BLANKS DETERMINATION OF THE IMPURITIES DETECTABLE BY HPLC HPLC-UV methods for purity assessment HPLC-UV-MS method for purity assessment LC-MS methods for purity assessment Impact of the purity of the calibrants on the value assignment of T 4 mass fraction in Laboratories B, C and D DETERMINATION OF THE WATER MASS FRACTION DETERMINATION OF THE ETHANOL MASS FRACTION Method Results DETERMINATION OF THE RESIDUAL ASH MASS FRACTION Experimental Results and discussion CALCULATION OF THE CERTIFIED VALUE FOR TOTAL T 4 MASS FRACTION CALCULATION OF THE INDICATIVE VALUE FOR T 3 MASS FRACTION EVALUATION OF UNCERTAINTIES AND CERTIFIED VALUES UNCERTAINTY EVALUATION Conceptual considerations Uncertainty source homogeneity Uncertainty source stability Uncertainty source batch characterisation Uncertainty source unknown impurity Uncertainty budget CERTIFIED VALUES METROLOGICAL TRACEABILITY

7 8. INSTRUCTIONS FOR USE TRANSPORT AND STORAGE MINIMUM SAMPLE INTAKE HYGROSCOPICITY DISSOLUTION USE OF THE CERTIFIED VALUES REFERENCES ACKNOWLEDGEMENTS ANNEX A - SHORT-TERM STABILITY STUDY: EXPERIMENTAL CONDITIONS AND EXAMPLES OF HPLC-UV CHROMATOGRAMS ANNEX B SHORT-TERM STABILITY STUDY: RAW DATA OF HPLC-UV ANALYSIS OF IRMM- 468 (2 SAMPLES, 3 REPLICATES PER SAMPLE) ANNEX C LONG-TERM STABILITY STUDY: EXPERIMENTAL CONDITIONS AND EXAMPLES OF HPLC-UV CHROMATOGRAMS ANNEX D LONG-TERM STABILITY STUDY: RAW DATA OF HPLC-UV ANALYSIS OF IRMM ANNEX E DETERMINATION OF THE IMPURITIES BY HPLC IN IRMM ANNEX F - RESULTS OF THE WATER MASS FRACTION DETERMINATION OF IRMM-468 MATERIAL BY COULOMETRIC KARL FISCHER TITRATION (LABORATORY C) ANNEX G DETERMINATION OF THE ETHANOL MASS FRACTION OF IRMM-468 (LABORATORY F)

8 GLOSSARY ANOVA a w b BAM CKFT CRM DAD DIT Analysis of variance water activity slope of the regression line Bundesanstalt für Materialforschung und prüfung (DE) Coulometric Karl Fischer Titration Certified Reference Material Photodiode Array Detector 3,5-diiodotyrosine ESI+ Electrospray Ionisation (positive mode) ε molar absorptivity F Snedecor F F crit critical value of Snedecor F Fig. figure FT 3 Free 3,3,5-triiodothyronine FT 4 Free thyroxine FT-IR Fourier Transform-Infra Red GC Gas Chromatography HPLC High Performance Liquid Chromatography ISO International Organization for Standardization IRMM Institute for Reference Materials and Measurements IVD In-Vitro Diagnostic k coverage factor LC Liquid Chromatography LGC LGC Limited (GB) LOD Limit of Detection LOQ Limit of Quantification m ep endpoint mass m s mass of the sample MIT 3-iodotyrosine (mono-iodotyrosine) MS Mass Spectrometry MS between Mean of Squares between groups MS q Mean of Squares MS within m/z n N ע NIST NMR Mean of Squares within groups mass to charge number ratio number of replicates number of vials degree of freedom National Institute of Standards and Technology (US) Nuclear Magnetic Resonance PTB Physikalisch-Technische Bundesanstalt (DE) PTFE PolyTetraFluoroEthylene R² coefficient of determination RM Reference Material RP Reversed-Phase RSD Relative Standard Deviation RT Retention Time s standard deviation s bb random variation between units SI Système International d Unités SIM Single Ion Monitoring S/N Signal to Noise ratio SS Sum of Squares t 0.05, ע T 2 T 3 T 4 T 4 w HPLC t of Student test at 95 % of confidence and ע degree of freedom 3,5-diiodothyronine 3,3,5-triiodothyronine thyroxine or 3,3,5,5 -tetraiodothyronine T 4 mass fraction as determined by HPLC T 4 w* HPLC T 4 mass fraction as determined by HPLC and corrected for the impurities which were detected in HPLC by only some of the laboratories T 4 w total total T 4 mass fraction taking into account the impurities detectable in HPLC as well as the ethanol, water and residual ash. TETRAC 3,3,5,5 -tetraiodothyroacetic acid 5

9 Temp. TRIAC TSH TSP temperature 3,3,5-triiodothyroacetic acid thyrotropin (thyroid stimulating hormone) Trimethylsilyl-tetradeuteropropionic acid sodium salt TT 3 Total concentration of 3,3,5- triiodothyronine in blood, equal to the sum of the free form and the proteinbound form of 3,3,5-triiodothyronine TT 4 Total concentration of thyroxine in blood, equal to the sum of the free form and the protein-bound form of thyroxine u b standard uncertainty of the slope u bb relative uncertainty component from homogeneity u * bb u c relative uncertainty of the maximum hidden between bottle heterogeneity combined standard uncertainty of the certified value and the measurement result u char relative uncertainty component from batch characterisation u CRM standard uncertainty of the certified value U CRM expanded uncertainty of the certified value expressed with a coverage factor k = 2, corresponding to a level of confidence of about 95% u lts relative uncertainty component from long-term stability u m measurement standard uncertainty of the result u storage 4 C relative uncertainty component from storage at 4 C u sts relative uncertainty component from short-term stability u unknown impurity relative uncertainty component from unknown impurity UV Ultra Violet X average 6

10 1. INTRODUCTION 1.1 Background: need for the CRM Thyrotropin (TSH), thyroxine (3,3,5,5 -tetraiodothyronine or T 4 ) and 3,3,5-triiodothyronine (T 3 ) play a major role in the diagnosis and monitoring of thyroid diseases. T 4 and T 3 are the primary hormones secreted by the thyroid gland. Their secretion is regulated by feedback from the hypothalamus and TSH from the pituitary gland. They are of major importance in the growth, development and sexual maturation of the human body and increase the energy expenditure in tissues. They are also involved in the regulation of the heart rhythm, body mass and metabolism. Therefore, their analysis is very frequently requested in endocrinology. In the blood, T 3 and T 4 occur in a free form (FT 3, FT 4 ) and in a protein-bound form. The sum of bound and free hormones is measured as the total hormone concentration, i.e. TT 3, TT 4. Typical physiological concentrations of TT 4 and TT 3 in adults are nmol/l and nmol/l, respectively [2]. The values of TT 4 and FT 4 concentrations in serum give an indication of the functional state of the thyroid gland. Hypothyroidism (thyroid gland deficiency) or hyperthyroidism (excessive hormone secretion) can be diagnosed and abnormal findings on physical examination of the thyroid gland can be interpreted after determination of the TT 4 and FT 4 concentrations. In order to achieve comparability of results from different analytical procedures and commercial diagnostic kits, standardisation is required using reference procedures and Certified Reference Materials (CRMs) as well as quality control materials. The Directive 98/79/EC is especially aiming to ensure the quality of In-Vitro Diagnostic (IVD) medical devices through the use of higher order reference materials and methods for calibration. In this context, a new higher order reference material for TT 4 and FT 4, IRMM-468, has been produced. It can be used by IVD manufacturers and reference laboratories for calibration of measurements procedures based on chromatographic techniques. The structure of thyroxine is shown in Figure 1. THYROXINE [ ] Figure 1 - Structure of thyroxine and CAS-number 7

11 1.2 Choice of the material to be certified From three commercially available materials the thyroxine provided by Acros Organics was selected because it appeared to be the most pure using HPLC-MS, HPLC-UV and GC-MS in scan mode. Thyroxine crystalline powder was obtained from Acros Organics (Geel, Belgium; product number: 21566, lot number: A ) with a claimed purity > 98.5 % (HPLC purity according to Acros Organics). 1.3 Design of the project The thyroxine material was aliquoted at IRMM in 100 mg units. The material was tested for homogeneity and stability (short- and long-term) and was characterised (identification and purity assessment) in five different laboratories with independent methods focusing on most likely occurring impurities. The characterisation was achieved in two steps: First, the peak area fraction in percent of T 4 and the impurities detectable by HPLC were determined by HPLC-UV, HPLC-UV-MS and HPLC-MS. The T 4 mass fraction was calculated in each laboratory by comparing the T 4 peak area to the peak areas of the impurities detectable by HPLC. The sum of all peak areas was considered as 100 %. Two approaches were used in order to calculate T 4 mass fraction from the HPLC results: 1. In the long-term stability study as well as in two laboratories participating to the characterisation study, the peak area fraction in percent of T 4 and the impurities were assumed to correspond to the respective mass fraction in percent. The mass fraction of T 4 as determined by HPLC (T 4 w HPLC ) was calculated as : T 4 w HPLC (T 4 peak area / Sum of peak areas for T 4 and HPLC-detectable impurities) x 100 % Note: For the measurements performed using UV detection, the Beer-Lambert s law should be considered: A = ε l c where ε is the molar absorption coefficient (L/(mol cm)); l is the path length (cm) and c is the concentration (mol/l). Therefore, a correction factor taking into account the molar mass of the molecules should be applied to estimate mass fractions from peak area fractions determined in UV. However in practice, the difference in peak areas before and after application of the correction factor is lower than the uncertainty of the certified value for T 4 mass fraction. The impact of the molar absorptivity on the calculation of T 4 mass fraction in IRMM-468 can be considered as negligible. 8

12 2. In the homogeneity and short-term stability studies as well as in three laboratories participating to the characterisation study, the measuring system for known impurities was calibrated using commercially available calibrants and was related to the mass of IRMM-468 analysed. The mass fraction of T 4 as determined by HPLC (T 4 w HPLC ) was calculated as: T 4 w HPLC = (100 % Sum of mass fractions of impurities) Note: The use of calibration curves to quantify the HPLC-detectable impurities in IRMM-468 implies to take into account the purity of the calibrants. However, as no reference material was available to calibrate the measuring system for the known impurities in IRMM-468, commercially available calibrants with a described purity 95 % were used. As described in Section 5.3.4, due to the very low mass fraction of the impurities determined in HPLC in IRMM-468, the impact of the calibrants purity on the determination of T 4 mass fraction was found to be negligible compared to the uncertainty of the the certified value. Both approaches used in the characterisation study of IRMM-468 provided similar results. The final value is the average of the averages of the mass fractions of T 4 determined by HPLC in the five laboratories. Second, the mass fractions of impurities non detectable by HPLC, i.e. ethanol, water and residual ash, were determined by NMR, CKFT and ashing, respectively. The mass fractions of these impurities were then substracted from the average T 4 mass fraction determined by HPLC to lead to the certified T 4 mass fraction in IRMM-468 (T 4 w total ). Studies and investigations were mainly performed in the frame of the Shared Cost Action IVD Thyroid G6RD-CT

13 2. PARTICIPANTS 2.1 Stability and homogeneity studies - Institute for Reference Materials and Measurements (IRMM), Geel, BE 2.2 Characterisation measurements The laboratories who participated to the characterisation study of IRMM-468 are: - Institute for Reference Materials and Measurements (IRMM), Geel, BE: two laboratories. - Physikalisch-Technische Bundesanstalt (PTB), Braunschweig, DE. - National Institute of Standards and Technology (NIST), Gaithersburg, US. - LGC Limited (LGC), Teddington, GB. - Bundesanstalt für Materialforschung und prüfung (BAM), Berlin, DE. In order to preserve the anonymity of the laboratories, they will be represented by a code, between A and F, in the certification report. The characterisation of IRMM-468 consisted of four different determinations: - The determination of impurities detectable by HPLC was performed by laboratory A, B, C, D and E. - The water mass fraction determination was carried out by laboratory B. - The ethanol mass fraction determination was achieved by laboratory F. - The residual ash determination was performed by laboratory E. 10

14 3. PREPARATION OF THE MATERIAL 3.1 Bottling The bottling was done at IRMM. A total number of 917 units were produced. Each unit consisted of an amber glass vial with approximately 100 mg of T 4. The bottling was carried out in a glove box under nitrogen gas. The vials were closed using a polytetrafluoroethylene (PTFE) coated rubber stopper. The outside surface of the vials was cleaned with a soft cloth. The vials were sealed using an aluminium cap. The main stock is stored between -40 and -70 C. 3.2 Dispatching of samples Samples for homogeneity and stability studies and for characterisation measurements were dispatched to participating laboratories using a courier service. The samples were shipped at 4 C and delivered within 24 hours. 11

15 4. TESTING OF THE MATERIAL 4.1 Homogeneity study The design of the short-term stability study enabled also the assessment of the homogeneity of the material. Considering the poor stability of IRMM-468 at 60 C (see Section ), only the data obtained at -70, -20 and 4 C were used for this assessment. In total, ten samples were analysed in triplicate using HPLC-UV. The sample numbers were randomly spread over the whole batch. A one-way ANOVA was carried out grouping the data by sample number (group = sample number). The results presented in Table 1 and expressed in mass fractions show that no significant difference was observed between the samples at temperatures where the material is stable (F < F crit ). Table 1. One-way ANOVA for the homogeneity study of IRMM-468 (number of replicates = 3). SUMMARY Temperature Storage time Group (=sample number) Average mass fraction Variance of the mass fraction [ C] [weeks] [% ] [%²] Average ( X ) ANOVA Source of Variation SS ע MS q F F crit Between Groups Within Groups Total

16 As MS between (0.04) < MS within (0.06), the random variation between units (s bb ) can not be estimated properly. Therefore, the maximum hidden between bottle heterogeneity (u* bb ) was evaluated as follows: u MS * within = 4 bb n N 1 2 ( n ) where N = number of vials, n = number of replicates. A u* bb of % was obtained. The individual sample averages were calculated as well as the average of all samples (99.54 %) (Table 1). With the exception of sample n 491 (-20 C, 2 weeks) where only two replicates were performed (n =2) due to experimental error in the laboratory, the individual averages of the other samples were found to be included in the interval determined by: average of all samples ± distance from value to limit (k = 2) which corresponds to X ± U CRM (k = 2) The material can thus be considered to be homogeneous at the level of the sample intake used in the HPLC-UV procedure for homogeneity testing of the material. The sample intake in that procedure being 10 mg, the material can be considered to be homogeneous at 10 mg sample intake level. 4.2 Stability study Short-term and long-term stability studies were performed. Both studies were carried out at IRMM following an isochronous set-up [3] that consists of the simultaneous analysis of reference and test samples. A defined number of samples was exposed for different periods of time to given temperatures, then brought back to the reference temperature of 70 C where no impact on the analyte content over a long time period is expected. At the end of the study, all samples were analysed for their impurities contents in one experimental run. The IRMM-468 samples were analysed by methods employing high performance liquid chromatography coupled to an ultra-violet detector (HPLC-UV). Very similar methods were used for short-term and long-term stability studies. The experimental conditions are summarised in Table 2. More details about the instrumentation, solution preparation, Limits of Detection and Quantification (LODs and LOQs) and calibration curves are presented in Annexes A and C for the short- and long-term stability, respectively. 13

17 Table 2. HPLC-UV experimental conditions for the stability study of IRMM-468 Stability study Short-term [4] Long-term HPLC column HPLC mobile phases HPLC gradient - C18 Supelco Discovery, 250 x 4.6 mm, 5 µm - Supelguard Discovery 18 guard column 20 x 4.0 mm, 5 µm A = 0.1 % diluted phosphoric acid/ acetonitrile (65+35; volume fractions) B = demineralised water Gradient: from 70 % of A and 30 % of B to 100 % of A until 5 min, held till 30 min. Flow rate = 1.0 ml/min for 5 min; 1.5 ml/min for 25 min. idem idem Temperature 25 C idem Injection volume 100 µl idem Detection 225 nm idem Gradient: from 50 % of A and 50 % of B to 100 % of A until 5 min, held till 17 min. Flow rate = 1.0 ml/min for 5 min; 1.5 ml/min for 17 min Short-term stability study Design of the short-term stability study The design of the short-term stability study is illustrated in Table 3. The study was conducted over a period of 4 weeks at 4 different temperatures (reference temperature = -70 C). Two IRMM-468 samples were analysed per time point and temperature. Each sample was analysed in triplicate. Expected organic impurities are 3-monoiodotyrosine (MIT), 3,5-diiodotyrosine (DIT), 3,5- diiodothyronine (T 2 ) and 3,3,5-triiodothyronine (T 3 ). A calibration curve was established for each impurity using commercially available calibrants from Sigma-Aldrich. The stability of IRMM-468 samples was expressed in terms of mass fraction of thyroxine (T 4 ) calculated as the difference between 100 % and the sum of the mass fractions of the impurities. Only impurities giving a signal higher than the LOQ were taken into account for the quantification of T 4. 14

18 Table 3. Sample numbers of IRMM-468 analysed in short-term stability study Sample number Temperature [ C] Time [weeks] (reference) (reference) Results of the short-term stability study Examples of HPLC chromatograms of a blank sample (methanol) (Fig. 1), calibrant mixture with 200 mg/l of each MIT, DIT, T 2, T 3 and T 4 (Figure 2) and IRMM-468 (Figure 3) sample are shown in Annex A. LODs and LOQs are presented in Annex A. They were estimated using calibration curves. No LOQ was determined for DIT. However, taking into account the similarity between DIT and T 2, the same LOQ can be used for DIT without reasonable doubt. As LODs and LOQs were given in terms of mass concentrations in mg/l, the peak area fractions of the impurities measured in IRMM-468, assumed to correspond to the mass fractions of these impurities, were converted into mass concentrations in mg/l for evaluation. Taking into account that the short-term stability was determined on IRMM-468 solutions at 200 mg/l, the traces of the different impurities expressed as mass fraction percentages were translated into mass concentrations using the following equation: Impurity concentration [mg/l] = impurity mass fraction 200 [mg/l] No MIT could be detected in IRMM-468. Traces of DIT could occasionally be detected at levels above the LOQ. T 2 was detected at levels above LOQ, between 2.5 and 5.1 %, in the samples n 473 and 482 stored at 60 C for 2 and 4 weeks, respectively. However no T 2 was detected in the samples n 23 and 32 also stored at 60 C for 2 and 4 weeks, respectively. 15

19 T 3 was detected as a constant impurity in IRMM-468 at levels above the LOQ. The mass fractions of DIT, T 2 and T 3 were calculated using the ratio of the peak areas obtained for the respective impurities in IRMM-468 and in the calibration curve. This was related to the exact initial mass of IRMM-468, used in the preparation of the sample solution, to finally yield the mass fraction of the impurity in IRMM-468. The mass fraction of T 4 was obtained as the difference between 100 % and the sum of DIT, T 2 and T 3 mass fractions. The mass fractions of T 4 in IRMM-468 short-term stability samples are summarised in Annex B. Data were evaluated with respect to the repeatability of the measurements within a sample (3 different readings of every sample) and to the repeatability of the measurements between the different samples from different temperature/time conditions. As illustrated in Figure 2, a regression line was calculated at each tested temperature by plotting T 4 mass fraction versus the storage time. Figure 2. Regression lines obtained at different storage temperatures for short-term stability study of IRMM-468 A) -20 C 100 T4 mass fraction y = 0.017x R 2 = Time [weeks] 16

20 B) 4 C 100 T4 mass fraction y = 0.038x R 2 = Time [weeks] C) 60 C 100 T4 mass fraction y = x R 2 = Time [weeks] The slope (b) of the regression line was tested for significance using the t-test. The slope is not significantly different from zero if the ratio of the slope and the uncertainty of the slope (u b ) is lower than the t-value at 95 % of confidence. Although a degradation of some samples at 60 C is obvious (see Figure 2 C), all slopes obtained at the different temperatures (Table 4) are not significantly different from zero (b/u b < t ע, 0.05 ). Due to the large scatter of results of the samples stored at 60 C, the uncertainty of 17

21 the slope observed is high, thus not leading to a significant outcome of the t-test. The observed repeatability of samples stored at -20 and 4 C corresponds to the expected repeatability of the HPLC measurements. Table 4. Test for significance of the slope: slopes (b), uncertainties (u b ) and b/u b of shortterm stability study for IRMM-468 Temperature b [ C] [week -1 ] Short-term stability study u b [week -1 ] b/u b ע, 0.05 t Thus, the stability of IRMM-468 could be demonstrated for a period of 4 weeks at 4 and -20 C whereas at 60 C the material shows clear signs of degradation. Therefore, the cooled dispatch of IRMM-468 samples at 4 C is required Long-term stability study Design of the long-term stability study The long-term stability study was conducted over a period of 12 months at 3 different temperatures (reference temperature = -70 C) as described in Table 5. Two samples were analysed per time point and temperature. Each sample was analysed in triplicate. The presence of MIT, T 2 and T 3 impurities was investigated. No calibration curve was established. The purity of IRMM-468 in long-term stability samples was determined by considering the sum of peak areas of T 4 and the impurities detected in HPLC-UV as 100 %. The peak area fractions of T 4 and the impurities were assumed to correspond to the respective mass fractions. The final results of the stability study were evaluated by regression analysis plotting the individual results against the storage time at the test temperature. 18

22 Table 5. Sample numbers of IRMM-468 analysed in long-term stability study Sample number Temperature [ C] Time [months] (reference) (reference) Results of the long-term stability study Annex C shows HPLC chromatograms of the blank sample (dissolution solvent used for the calibration solutions) (Figure 1), calibrant mixture with 50 mg/l of MIT, T 2, T 3 and T 4 (Figure 2) and IRMM-468 (Figure 3) sample. The purity of IRMM-468 material was calculated only taking impurities above the LOQ into consideration. The LOQ was determined as the mass fraction giving a signal-to-noise ratio (S/N) equal to 10 (S/N=10). LODs and LOQs of MIT, T 2, T 3 and T 4 are presented in Annex C. T 3 impurity was quantified in IRMM-468 at levels equal or higher than the LOQ. No MIT and no T 2 were detected. The mass fractions of T 4 in IRMM-468 samples are presented in Annex D. A one-way ANOVA and a trend analysis were performed on these results. The ANOVA of the data, comparing the results grouped by temperature, is presented in Table 6. No significant difference between the T 4 mass fractions in the samples stored at the reference temperature (-70 C) and the test temperatures -20 C and 4 C, respectively, was observed (F < F crit ). 19

23 Table 6. One-way ANOVA of the long-term stability data for IRMM-468 SUMMARY Groups (= storage temperature) Number of replicates Average mass fraction Variance of the mass fraction [ C] [%²] ANOVA Source of Variation SS ע MS q F F crit Between Groups Within Groups Total As illustrated in Figure 3, a trend analysis was performed by plotting the T 4 mass fraction of IRMM-468 samples versus storage time and by calculating a regression line. Figure 3. Regression lines obtained at different storage temperatures for long-term stability study of IRMM-468 (2 samples per time, 3 replicates per sample) A) -20 C 100 T 4 mass fraction y = x R 2 = Time [months] 20

24 B) 4 C 100 T 4 mass fraction y = x R 2 = Time [months] The slope of the regression line was then tested for significance. Results are presented in Table 7. Table 7. Test for significance of the slope: slopes (b), uncertainties (u b ) and b/u b of long-term stability study for IRMM-468 Long-term stability study Temperature [ C] b [months -1 ] u b b/u b ע, 0.05 t [months -1 ] No significant trend was observed at -20 C over 12 months indicating a good stability of IRMM-468 at this temperature. A shelf-life of 3 years was determined with u lts set at 0.2 %. However, at 4 C, a significant slope was obtained over time showing a degradation of IRMM-468 between 6 and 12 months. No significant slope was observed from the data between 0 and 6 months at 4 C. In order to maintain a long-term stability of the main batch, the IRMM-468 samples will be stored at IRMM between -40 and -70 C. As the validity of the certificate is 6 months after purchase, the vial will be stored between -20 and -70 C on receipt by the customer. Taking into account the intermediate storage of IRMM-468 samples at 4 C during the certification period (25 months), a standard uncertainty contribution (0.14 %) corresponding to the period of storage at 4 C was added to the uncertainty budget (Table 22). 21

25 5. CHARACTERISATION MEASUREMENTS As indicated under Section 2.2, the characterisation aiming at the detection of impurities by HPLC was performed through an intercomparison between five laboratories. Three different characterisation techniques were used: HPLC-UV in laboratory A, B and C; HPLC-UV-MS in laboratory D; HPLC-MS in laboratory E. As for the stability study, IRMM-468 was characterised in terms of purity (peak areafraction). In addition, the water, ethanol and residual ash mass fractions, respectively, of IRMM-468 were determined by three laboratories: Laboratory C, E and F. The analytical methods used for the characterisation are described in detail in Section 5.3. Each laboratory used its own optimised procedure for sample preparation, method of injection, chromatographic separation and detection. All procedures and measurements were recorded and reported by each laboratory. A minimum of 3 replicates per sample (in total 6 samples) was analysed for the HPLC characterisation. 5.1 Calibrants Each laboratory prepared separate calibration solutions from commercially available DIT, MIT, T 2, T 3 and T 4. The calibrants were from Sigma-Aldrich, Fluka and Acros. Their purity was 95 % (Table 8).The calibrants were used to identify T 4 and the impurities. They were also used in the characterisation study to build calibration curves in order to check the linearity of the detector response and to estimate LODs and LOQs. In some laboratories, the peak area fractions obtained for the respective impurities in IRMM-468 and in the calibration curves were compared in order to estimate the mass fractions of the impurities in IRMM-468. The impact of using calibrants, which purity ( 95 %) is not certified, on the mass fraction of T 4 has been evaluated and was found to be negligible (see Section 5.3.4). Table 8. Purity of the calibrants used for the certification of IRMM-468, as specified by the supplier Sigma-Aldrich Fluka Acros T4 98 > 98.5 T3 95 T2 > DIT > 97.5 MIT > Analytical blanks Analytical blanks (consisting of HPLC mobile phase injections) were performed. All solvents used were LC grade. 22

26 5.3 Determination of the impurities detectable by HPLC This section presents the results obtained in each of the five laboratories. The experimental conditions of the different methods are summarised in Table 9. Additional information about the instrumentation used and LOD/LOQ is given in Annex E. As described in Section 1.2, the T 4 mass fraction was calculated in each laboratory by comparing the T 4 peak area to the sum of the peak areas of the impurities detectable by HPLC. The sum of all peak areas, including that of T 4, was considered as 100 %. Then two approaches were used in order to calculate T 4 mass fraction from the HPLC results: 1. In laboratories A and E, the peak area fractions of T 4 and of the impurities were assumed to correspond to the respective mass fractions in %. The mass fraction of T 4 as determined by HPLC (T 4 w HPLC ) was calculated as : T 4 w HPLC (T 4 peak area / Sum of peak areas for T 4 and HPLC-detectable impurities) x 100 %. The negligible influence of the different molar absorptivity of the studied compounds on the determination of T 4 mass fraction by HPLC-UV in Laboratory A is described in Section In laboratories B, C and D, the measuring system for known impurities was calibrated using commercially available calibrants and was related to the mass of IRMM-468 analysed. The mass fraction of T 4 as determined by HPLC (T 4 w HPLC ) was calculated as: T 4 w HPLC = (100 % Sum of mass fractions of impurities). The negligible impact of the calibrants purity on the determination of T 4 mass fraction by Laboratory B, C and D is demonstrated in Section For technical reasons, some laboratories could not investigate the presence of MIT and/or DIT impurities in IRMM-468. In these cases, the average value of MIT and/or DIT mass fraction obtained by the other laboratories was substracted from the T 4 mass fraction determined by HPLC. The resulting corrected average T 4 mass fractions (T 4 w* HPLC ) are given in Table 17. For laboratories which were able to determine MIT and DIT, the values provided by each laboratory were substracted from the T 4 mass fraction determined by HPLC, respectively. The average of the HPLC results obtained in each laboratory (average T 4 w HPLC ) was calculated and is presented in Table 17. Then the water, ethanol and residual ash mass fractions of IRMM-468 were determined as described under Sections 5.4, 5.5 and 5.6, respectively. Finally, the combined standard uncertainty of the result was calculated and will be described in Section 6. 23

27 Table 9. HPLC measurement conditions Laboratory Method HPLC column HPLC mobile phases A HPLC-UV Hypersil C x 2.1 mm B HPLC-UV C18 ProntoSIL analytical column 250 x 4.6 mm, 5 µm [volume fraction] 50 % Methanol - 50 % diluted formic acid (0.1 %; v/v) 70 % Methanol 30 % diluted phosphoric acid (0.1 %) Elution conditions Isocratic; flow rate = 0.2 ml/min Isocratic; flow rate = 0.7 ml/min Temperature [ C] Injection volume [µl] nm nm Detection C HPLC-UV - C18 Supelco Discovery, 250 x 4.6 mm, 5 µm - Guard column Discovery 20 x 4.0 mm, 5 µm A = 0.1 % diluted phosphoric acid / acetonitrile (65+35) B = demineralised water Gradient [volume fractions]: from 70 % of A and 30 % of B to 100 % of A until 5 min, held till 30 min Flow rate = 1.0 ml/min for 5 min; 1.5 ml/min for 25 min nm D HPLC-UV- MS Phenomenex Hypersil 3 C18, 150 x 2 mm, 3 µm E HPLC-MS Zorbax Eclipse XDB- C18 column 2.1 x 150 mm 20 % diluted formic acid (0.1 %) 80 % methanol containing 0.1 % formic acid A = 0.1 % diluted acetic acid B = 0.1 % acetic acid in methanol Isocratic; flow-rate = 0.2 ml/min Gradient [volume fractions]: 0 min = 35 % B; 5 min = 40 % B; 10 min = 55 % B; 35 min = 55 % B; 40 min = 90 % B; 45 min = 35 % B Flow rate = 0.25 ml/min UV: 254 nm - ESI+/MS* in SIM mode 25 5 ESI+/MS* in SIM mode 24

28 HPLC-UV methods for purity assessment Procedure applied by laboratory A One sample of IRMM-468 was analysed in triplicate. Spectra taken at the retention time of the chromatographic peaks and at different retention times were further examined for any evidence of impurities at other wavelengths. The chromatograms obtained for the T4 material in mobile phase were compared to the chromatograms obtained for the standard solution of commercially available T4 (Sigma). The spectrum obtained at the retention time of T4 in the chromatogram obtained for IRMM-468 was compared to the scan made by UV spectrophotometer. Additional qualitative purity profiling of IRMM-468 samples was performed by HPLC-ESI- MS. Reconstructed ion chromatograms of IRMM-468 showed ions corresponding to [M+H] + ions of T 2 (mass to charge ratio, m/z = 536), T 3 (m/z = 652) and T 4 (m/z = 778). Finally, the identity of T 4 in IRMM-468 was confirmed using Nuclear Magnetic Resonance (NMR): 1 H-NMR and 13 C-NMR spectroscopy. The purity of the sample was calculated from the peak areas obtained in UV and assuming that the peak area fractions correspond to the mass fractions i.e.: T 4 w HPLC (T 4 peak area / Sum of peak areas) 100 % The quantitative results are presented in Table 10. Table 10. Laboratory A: Mass fraction of T 4 in IRMM-468, expressed as an area fraction (1 sample, 3 replicates) Replicate number T 4 area fraction Average s

29 Note: As Laboratory A did not investigated MIT and DIT impurities, the average mass fraction of these two impurities obtained in the other laboratories (0.08 and %, respectively) was considered. The resulting T 4 mass fraction presented in Table 17 was %. Because of the Beer-Lambert s law: A = ε l c where A is the absorption; ε is the molar absorption coefficient (L/(mol cm)); l = path length (cm); c = concentration (mol/l) a correction factor, taking into account the molar mass of the compound, should be applied to estimate mass fractions from peak area fractions. This correction factor can be expressed as follows: peak area fraction of the impurity x molar mass of the impurity [kg/mol] molar mass of T 4 [kg/mol] The mass fraction of T 4 was re-calculated using the corrected mass fractions of the impurities. The resulting T 4 mass fraction was %. The difference between the corrected and the non-corrected T 4 mass fraction is 0.12 % and is much lower than the uncertainty of the certified value for T 4 mass fraction 0.7 % determined in section 6. Therefore, the influence of the Beer-Lambert s law on the determination of T 4 mass fraction by HPLC-UV can be considered as negligible Procedures applied by laboratory B One IRMM-468 sample was used to prepare one stock solution. From the stock solution, 5 working solutions were prepared and analysed in triplicate. The 15 results were reported as a single result (average of the 15 injections). The whole procedure was repeated on three days, using new working solutions on each day. The 3 averaged results are summarised in Table 11. The presence of T 3, T 2 and MIT impurities in IRMM-468 was investigated in UV and MS detection. However, for their quantification, only individual UV peak areas were determined. Calibration curves were established in UV detection for T 3, T 2, MIT and as an additional information for T 4 using commercially available standards. The mass concentrations of T 3, T 2 and MIT in IRMM-468 were calculated for each using its ratio of the peak area obtained in IRMM-468 to the calibrator. This was related to the exact initial mass of IRMM-468 (T 4 ), used in the preparation of the sample solution, to finally yield the mass fraction of the impurity in IRMM-468. The mass fraction of T 4 was obtained by difference between 100 % and the sum of T 3, T 2 and MIT mass fractions in %. T 3, T 2 and MIT impurities were quantified in IRMM-468. One additional and unknown impurity, eluting 0.2 min before MIT, was detected in the UV chromatogram of IRMM-468 and each commercially available standard T 4, T 3, T 2 and MIT. The area fraction of this unknown impurity was estimated using the ratio of peak areas for the unknown impurity and for MIT, related to the percentage of MIT in IRMM-468. The unknown impurity represented 0.13 % of IRMM-468. This impurity could not be identified nor the LOQ determined. Therefore the mass fraction of this impurity was not taken into account to assess 26

30 the mass fraction of thyroxine in IRMM-468. However, a relative uncertainty contribution of 0.13 % was considered in the uncertainty budget of IRMM-468 (see Table 22). Table 11. Laboratory B: Purity of IRMM-468, expressed as a mass fraction (1 sample, measured on 3 days in 5 replicates): Day MIT T 2 T 3 T Average s Procedures applied by laboratory C One IRMM-468 sample was analysed in 10 replicates. The presence of MIT, T 2, T 3 impurities in IRMM-468 was investigated using the same HPLC-UV method as described for the short-term stability study (see Section ). Calibration curves were established for MIT, T 2, T 3 and as an additional information for T 4. Coefficients of determination (R²) and slopes of the curves are given in Annex E. A different slope (b) was observed for MIT (b = L/mg) compared to that of the other impurities (b between and L/mg). This could be related to the relatively low coefficient of determination obtained for MIT (R² = 0.862) or to the different chemical structure of MIT (tyrosine base molecule) and T 2, T 3, T 4 (thyronine base molecule) (Figure 4). 27

31 TYROSINE THYRONINE Figure 4 Structure of tyrosine and thyronine The results of the purity determination of IRMM-468 are presented in Table 12. Only T 3 was higher than LOQ and could be quantified as an impurity in the samples. The T 3 mass fraction was determined using the calibration curve and was related to the exact mass of IRMM-468 used to prepare the sample solution. The T 4 mass fraction was calculated by difference between 100 % and the T 3 mass fraction. Table 12. Laboratory C: Purity of IRMM-468, expressed as a mass fraction (1 sample, 10 replicates): T 3 T Average s

32 HPLC-UV-MS method for purity assessment Procedure applied by laboratory D IRMM-468 purity was investigated in two samples, each analysed in 10 replicates on three occasions. In addition to 3,5-diiodothyronine (T 2 ) and 3,3,5- triiodothyronine (T 3 ) impurities, the presence of intermediate products and by-products of synthesis of T 4 was checked. Possible target compounds were deduced from synthesis routes described in the literature [5-11]: 3,3,5,5 -tetraiodothyroacetic acid (TETRAC), 3,3,5-triiodothyro-acetic acid (TRIAC), 3,5-diiodothyroacetic acid, and 3,5-diiodotyrosine (DIT), tyrosine. A general check for impurities in T 4 material was also performed by GC-MS. No one of the intermediate products and by-products of synthesis substances except DIT could be detected by HPLC-MS and GC-MS. In addition, Fourier Transform-Infra Red (FT-IR) measurements were carried out. Spectra corresponded to expectations and the identification of T 4 was confirmed. Calibration solutions of commercially available T 2 and T 3 were prepared at the concentration expected in IRMM-468 which is a mass fraction about 0.01 % for T 2 and 0.3 % for T 3. Calibration solutions of commercially available DIT were also prepared. T 3 and T 4 measurements yielded both an MS and a UV based estimate of the respective mass fraction. No significant difference (Student s t-test, 5 % level) could be detected between the MS- and UV-based results. The results for T 3 and T 4 were the pooled mass fraction averages of the respective MS- and UV- results. In the case of DIT and T 2, only the MS results were considered as the UV-signal was lower than the detection limit. The mass fractions of T 2 and T 3 in IRMM-468 solution (averaged over the 10 runs) were calculated using the calibration curves and the initial mass of IRMM-468 sample. This was reported as a single measurement result. The same procedure was repeated on three different occasions. The three quantification results are compiled in Table 13. The mass fraction of T 4 among all components was obtained by difference between 100 % and the impurity mass fractions. The result of quantification of DIT by HPLC-MS was a mass fraction of % in IRMM-468. As a single measurement was carried out, no uncertainty statement can directly be derived from the repetition of measurements. Therefore a rectangular probability distribution was used between % and % as a rough estimate (half width of interval round the given value = %). A standard uncertainty of % for DIT quantification was then determined using the following equation [1]: u =

33 Table 13. Laboratory D: Purity of IRMM-468, expressed as a mass fraction (2 samples, 3*10 replicates): Sample T 3 T 3 Average T 3 T 2 T 2 DIT Calculated* number (MS) (UV) (MS&UV) (MS) (UV) T , ** 99, ** 99,67 Average s Sample T 3 T 3 Average T 3 T 2 T 2 DIT Calculated* number (MS) (UV) (MS&UV) (MS) (UV) T [% ] [% ] [% ] [% ] [% ] [% ] ** ** Average s * T 4 w HPLC = (100 % average T 3 (MS&UV) T 2 (MS) DIT (MS)) ** As a single measurement of DIT was performed, the same value of DIT mass fraction was used to calculate the mass fractions of T 4 in each replicate The average of T 4 mass fractions was calculated on both samples and was % with a standard deviation of 0.01 %, 30

34 LC-MS methods for purity assessment Procedure applied by laboratory E One IRMM-468 sample was analysed in triplicate. DIT, T 2, T 3 and reverse T 3 were the targeted impurities. Reverse T 3 (3,3',5'- triidodthyronine) is a biologically inactive isomer of T 3 [12] that can be separated from T 3 using reversed-phase liquid chromatography. A working solution of IRMM-468 was prepared at 100 mg/l. Calibration solutions of DIT, T 2, T 3, reverse T 3 and T 4 were prepared at the same concentration in order to estimate LODs and LOQs but were not used to quantify the mass fractions of the impurities. IRMM-468 was analysed preliminarily by full scan HPLC-MS and HPLC-MS-MS. However, due to the lack of sensitivity of quadrupole mass spectrometers operating in full scan mode, no impurity peaks were observed in the sample. A targeted analysis of possible and expected impurities in the sample (DIT, T 2, T 3, and reverse T 3 ) was carried out using HPLC-MS in the selected ion monitoring mode. No detectable T 2 and no reverse T 3 were found. DIT was detected at levels higher than LOD but lower than LOQ. Therefore DIT was not taken into account to calculate the mass fraction of T 4 in IRMM-468 but an uncertainty contribution related to DIT LOQ was added to the uncertainty budget of IRMM-468 (see Table 22). Finally T 3 was quantified as an impurity in IRMM-468 using the relative ion intensities of T 4 and T 3 in MS. The intensity fractions of T 4 and T 3 were assumed to correspond to the respective mass fractions. This approach does not take into consideration the differences in the electrospray ion yields of DIT and T 3 or T 4 (DIT contains one phenyl base molecule whereas T 3 and T 4 contain two phenyl base molecules). However, as only T 3 and T 4 were quantifiable and given their similar chemical structure, the probability of such a bias is rather low. The results of the purity assessment are presented in Table 14. Table 14. Laboratory E: Purity of IRMM-468, expressed as a mass fraction (1 sample, 3 replicates): T 4 DIT T 2 T 3 T 4 1 < LOQ < LOD < LOQ < LOD < LOQ < LOD Average s

35 Impact of the purity of the calibrants on the value assignment of T 4 mass fraction in Laboratories B, C and D As described in Table 8 (Section 5.1), the purity of the calibrants used for the determination of T 4 mass fraction in IRMM-468 was between 95 and more than 99 %. The impact of using calibrants not certified for their purity on the determination of T 4 mass fraction was evaluated by considering the less favourable case - where each calibrant is only 95 % pure (lowest purity percent described for the calibrants by the supplier) and - where the highest mass fraction of the HPLC-detectable impurities was observed by the laboratories. Table 15 presents the T 4 mass fraction obtained in the case of calibrants 100 % pure and 95 % pure, considering the maximum HPLC-detectable impurities mass fractions observed in the laboratories. The comparison of T 4 mass fraction obtained with 100 % pure calibrants and with 95 % pure calibrants shows a difference at the second digit after the coma only. The worst case of an eventual underestimation of T 4 mass fraction in IRMM-468 shows a bias from the certified value which is largely covered by the certified uncertainty of 0.7 %. Therefore, the impact of the calibrants purity can be considered as negligible in the case of IRMM-468. Table 15. Evaluation of the impact of calibrants purity on the determination of T 4 mass fraction IRMM-468 MIT DIT T 2 T 3 Maximum impurities mass fractions observed by the laboratories If the calibrants are 100 % pure: Corresponding minimum T 4 mass fraction = (100 % maximum impurities mass fractions) If the calibrants are 95 % pure: Bias: 5 % of maximum impurities mass fractions Resulting overestimation (105 %) of impurities mass fractions Resulting underestimated T 4 mass fraction = (100 % overestimated impurities mass fractions)