M10 B M
IOANALYTICAL  ETHOD 
V  
ALIDATION
 
 
 
 
 
 
 
 
 
This draft guidance, when finalized, will represent the current thinking of the Food and Drug 
Administration (FDA or Agency) on this topic.  It does not establish any rights for any person 
and is not binding on FDA or the public.  You can use an alternative approach if it satisfies the 
requirements of the applicable statutes and regulations.  To discuss an alternative approach, 
contact the FDA staff responsible for this guidance as listed on the title page.  The draft 
guidance has been left in the original International Council for Harmonisation format.  The 
final guidance will be reformatted and edited to conform with FDAs good guidance practice 
regulation and style. 
 
 
For questions regarding this draft document, contact (CDER) Brian Booth 301-796-
1508 or (CBER) Tsai-Lien Lin 240-402-8698. 
 
     
 
 
 
 
INTERNATIONAL  COUNCIL FOR HARMONISATION OF TECHNICAL 
REQUIREMENTS  FOR PHARMACEUTICALS FOR HUMAN USE 
 
 
ICH HARMONISED GUIDELINE 
 
 
B M V  
IOANALYTICAL  ETHOD  ALIDATION
M10 
 
 
Draft version  5.1   Step  1   
Endorsed on 7 June 2018 
Currently  under  public  consultation 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
At  Step 2 of the ICH Process, a consensus draft text or guideline, agreed by the 
appropriate ICH Expert Working Group, is transmitted by the ICH Assembly to the 
regulatory authorities of the  ICH  regions  for internal and external consultation, 
according to national or regional procedures. 
     
M10 
Document History 
 
Code  History  Date 
M10  Endorsement by the Members of the ICH Assembly  7 June 2018 
under  Step 2 and release for  public  consultation 
(document dated 20 December 2018). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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1  ICH HARMONISED GUIDELINE 
BIOANALYTICALMETHODVALIDATION 
2     
M10 
3 
4  ICH Consensus Guideline 
5   
6  TABLE OF CONTENTS 
7  1. INTRODUCTION..........................................................................................................3 
8  1.1 Objective ....................................................................................................................... 3 
9  1.2 Background ................................................................................................................... 3 
10  1.3 Scope............................................................................................................................. 3 
11  2. GENERAL PRINCIPLES..............................................................................................4 
12  2.1 Method Development ..................................................................................................... 4 
13  2.2 Method Validation .......................................................................................................... 5 
14  2.2.1 Full Validation     ............................................................................................... 5 
15  2.2.2 Partial Validation ................................................................................................ 6 
16  2.2.3 Cross Validation .................................................................................................. 6 
17  3. CHROMATOGRAPHY .................................................................................................6 
18  3.1 Reference Standards ....................................................................................................... 6 
19  3.2 Validation ...................................................................................................................... 7 
20  3.2.1 Selectivity ............................................................................................................ 7 
21  3.2.2 Specificity ............................................................................................................ 8 
22  3.2.3 Matrix Effect........................................................................................................ 9 
23  3.2.4 Calibration Curve and Range ............................................................................... 9 
24  3.2.5 Accuracy and Precision...................................................................................... 11 
25  3.2.5.1 Preparation of Quality Control Samples .......................................................... 11 
26  3.2.5.2 Evaluation of Accuracy and Precision.............................................................. 11 
27  3.2.6 Carry-over......................................................................................................... 12  ICH M10 Guideline 
 
 
28  3.2.7 Dilution Integrity ............................................................................................... 12 
29  3.2.8 Stability ............................................................................................................. 13 
30  3.2.9 Reinjection Reproducibility ................................................................................ 16 
31  3.3 Study Sample Analysis ................................................................................................. 16 
32  3.3.1 Analytical Run ................................................................................................... 16 
33  3.3.2 Acceptance Criteria for an Analytical Run .......................................................... 17 
34  3.3.3 Calibration Range ............................................................................................. 18 
35  3.3.4 Reanalysis of Study Samples............................................................................... 19 
36  3.3.5 Reinjection of Study Samples .............................................................................. 20 
37  3.3.6 Integration of Chromatograms ........................................................................... 21 
38  4. LIGAND BINDING ASSAYS ......................................................................................21 
39  4.1 Key Reagents ............................................................................................................... 21 
40  4.1.1 Reference Standard ............................................................................................ 21 
41  4.1.2 Critical Reagents ............................................................................................... 21 
42  4.2 Validation .................................................................................................................... 22 
43  4.2.1 Specificity .......................................................................................................... 22 
44  4.2.2 Selectivity .......................................................................................................... 23 
45  4.2.3 Calibration Curve and Range ............................................................................. 24 
46  4.2.4 Accuracy and Precision...................................................................................... 25 
47  4.2.4.1 Preparation of Quality Control Samples .......................................................... 25 
48  4.2.4.2 Evaluation of Accuracy and Precision.............................................................. 25 
49  4.2.5 Carry-over......................................................................................................... 26 
50  4.2.6 Dilution Linearity and Hook Effect ..................................................................... 26 
51  4.2.7 Stability ............................................................................................................. 27 
52  4.3 Study Sample Analysis ................................................................................................. 28 
53  4.3.1 Analytical Run ................................................................................................... 28 
54  4.3.2 Acceptance Criteria for an Analytical Run .......................................................... 28 
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55  4.3.3 Calibration Range ............................................................................................. 29 
56  4.3.4 Reanalysis of Study Samples............................................................................... 30 
57  5. INCURRED SAMPLE REANALYSIS   ......................................................................31 
58  6. PARTIAL AND CROSS VALIDATION.......................................................................33 
59  6.1 Partial Validation.......................................................................................................... 33 
60  6.2 Cross Validation ........................................................................................................... 34 
61  7. ADDITIONAL CONSIDERATIONS ...........................................................................35 
62  7.1 Analytes that are also Endogenous Compounds ............................................................. 35 
63  7.1.1 Quality Control Samples .................................................................................... 36 
64  7.1.2 Calibration Standards ........................................................................................ 37 
65  7.1.3 Selectivity, Recovery and Matrix Effects.............................................................. 37 
66  7.1.4 Parallelism ........................................................................................................ 38 
67  7.1.5 Accuracy and Precision...................................................................................... 38 
68  7.1.6 Stability ............................................................................................................. 38 
69  7.2 Parallelism ................................................................................................................... 39 
70  7.3 Recovery ..................................................................................................................... 39 
71  7.4 Minimum Required Dilution......................................................................................... 39 
72  7.5 Commercial and Diagnostic Kits .................................................................................. 40 
73  7.6 New or Alternative Technologies .................................................................................. 41 
74  7.6.1 Dried Matrix Methods........................................................................................ 41 
75  8. DOCUMENTATION....................................................................................................42 
76  8.1 Summary Information .................................................................................................. 42 
77  8.2 Documentation for Validation and Bioanalytical Reports ............................................... 43 
78  9. GLOSSARY .................................................................................................................51 
79   
80 
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81  1. INTRODUCTION 
82  1.1 Objective    
83  This guideline is intended to provide recommendations for the validation  of bioanalytical  assays 
84  for chemical and biological  drug quantification  and their application  in the analysis of study 
85  samples. Adherence to the principles  presented in this guideline  will  improve  the quality  and 
86  consistency of the bioanalytical  data in support of the development and market approval of both 
87  chemical and biological  drugs. 
88  The objective of the validation  of a bioanalytical  assay is to demonstrate that it is suitable for 
89  its intended purpose. Changes from the recommendations  in this guideline  may be acceptable 
90  if  appropriate  scientific  justification  is provided.  Applicants  are encouraged  to consult  the 
91  regulatory authority(ies)  regarding significant  changes in method validation  approaches when 
92  an alternate approach is proposed or taken. 
93  1.2 Background    
94  Concentration  measurements  of chemical  and biological  drug(s)  and their  metabolite(s)  in 
95  biological  matrices  are an important  aspect of  drug  development.  The  results  of  pivotal 
96  nonclinical  toxicokinetic  (TK)/pharmacokinetic  (PK) studies and of clinical  trials,  including 
97  comparative  bioavailability/  bioequivalence  (BA/BE) studies,  are used to make regulatory 
98  decisions  regarding  the safety and efficacy of drug products. It is therefore critical  that the 
99  bioanalytical  methods used are well characterised, appropriately  validated and documented in 
100  order to ensure reliable data to support regulatory decisions.   
101  1.3 Scope    
102  This guideline  describes the method validation  that is expected for bioanalytical  assays that are 
103  submitted  to support regulatory submissions.  The guideline  is applicable  to the validation  of 
104  bioanalytical  methods used to measure concentrations of chemical and biological  drug(s) and 
105  their  metabolite(s)  in  biological  samples  (e.g., blood,  plasma,  serum,  other body  fluids  or 
106  tissues)  obtained  in  pivotal  nonclinical  TK/PK studies  that  are used  to  make  regulatory 
107  decisions and all phases of clinical  trials in regulatory submissions.  Full  method validation  is 
108  expected for the primary matrix(ces) intended  to support regulatory  submissions.  Additional 
109  matrices should  be partially  validated  as necessary. The analytes that should  be measured in 
110  nonclinical  and clinical  studies  and the types of studies  necessary to support  a regulatory 
111  submission  are described in other ICH and regional regulatory documents. 
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112  For studies that are not submitted  for regulatory  approval  or not considered  for regulatory 
113  decisions  regarding  safety, efficacy or labelling  (e.g., exploratory  investigations),  applicants 
114  may decide on the level of qualification  that supports their own internal decision making.   
115  The information  in this guideline  applies to the quantitative  analysis by ligand  binding  assays 
116  (LBAs)  and  chromatographic  methods  such  as  liquid  chromatography  (LC)  or  gas 
117  chromatography (GC), which are typically  used in combination  with mass spectrometry (MS) 
118  detection and occasionally  with other detectors. 
119  For studies that are subject to Good Laboratory Practice (GLP) or Good Clinical Practice (GCP) 
120  the bioanalysis  of study samples should also conform to their requirements. 
121  The  bioanalysis  of  biomarkers  and  bioanalytical  methods  used  for  the  assessment  of 
122  immunogenicity  are not within the scope of this guideline. 
123  2. GENERAL PRINCIPLES 
124  2.1 Method Development     
125  The purpose of bioanalytical  method development is to define the design, operating conditions, 
126  limitations  and suitability  of the method for its intended purpose and to ensure that the method 
127  is optimised  for validation. 
128  Before the development of a bioanalytical  method, the applicant should understand the analyte 
129  of interest (e.g., the physicochemical  properties of the drug, in vitro and in vivo metabolism and 
130  protein binding)  and consider aspects of any prior analytical methods that may be applicable. 
131  Method  development  involves  optimising  the  procedures  and  conditions  involved  with 
132  extracting and detecting the analyte. Method development can include  the optimisation  of the 
133  following  bioanalytical  parameters to ensure that the method is suitable for validation: 
134    Reference standards 
135    Critical reagents 
136    Calibration  curve 
137    Quality control samples (QCs) 
138    Selectivity  and specificity 
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139    Sensitivity 
140    Accuracy 
141    Precision 
142    Recovery   
143    Stability  of the analyte in the matrix 
144    Minimum  Required Dilution  (MRD) 
145  Bioanalytical  method  development  does not  require  extensive  record  keeping  or notation. 
146  However, the applicant should record the changes to procedures as well as any issues and their 
147  resolutions to provide a rationale for any changes made to validated methods immediately  prior 
148  to or in the course of analysing  study samples for pivotal  studies. 
149  Once the method has been developed, bioanalytical  method validation  proves that the optimised 
150  method is suited to the analysis of the study samples.   
151  2.2 Method Validation  
152  2.2.1 Full Validation     
153  Bioanalytical  method validation  is essential to ensure the acceptability  of assay performance 
154  and the reliability  of analytical results. A bioanalytical  method is defined as a set of procedures 
155  used  for  measuring  analyte  concentrations  in  biological  samples.  A  full  validation  of  a 
156  bioanalytical  method should  be performed when establishing  a bioanalytical  method  for the 
157  quantification  of an analyte in clinical  and in pivotal nonclinical  studies. Full validation  should 
158  also be performed when implementing  an analytical method that is reported in the literature and 
159  when a commercial kit is repurposed for bioanalytical  use in drug development.  Usually one 
160  analyte has to be determined, but on occasion it may be appropriate to measure more than one 
161  analyte.  This  may involve  two different  drugs,  a parent drug  with  its  metabolites  or  the 
162  enantiomers or isomers of a drug. In these cases, the principles of validation  and analysis apply 
163  to all analytes of interest. 
164  For  chromatographic  methods  a  full  validation  should  include  the  following  elements: 
165  selectivity,  specificity (if necessary), matrix effect, calibration  curve (response function),  range 
166  (lower limit  of quantification  (LLOQ) to upper limit  of quantification  (ULOQ)), accuracy, 
167  precision, carry-over, dilution  integrity,  stability  and reinjection  reproducibility.   
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168  For LBAs the following  elements should be evaluated: specificity, selectivity,  calibration  curve 
169  (response function),  range (LLOQ to ULOQ), accuracy, precision,  carry-over (if necessary), 
170  dilution  linearity,  parallelism  (if necessary, conducted during sample analysis) and stability.   
171  The matrix used for analytical method validation  should be the same as the matrix of the study 
172  samples, including  anticoagulants  and additives. In some cases, it may be difficult  to obtain an 
173  identical  matrix to that of the study samples (e.g., rare matrices such as tissue, cerebrospinal 
174  fluid, bile). In such cases surrogate matrices may be acceptable for analytical method validation. 
175  The surrogate matrix  should  be selected and justified  scientifically  for use in the analytical 
176  method.   
177  A specific,  detailed,  written description  of the bioanalytical  method should  be established  a 
178  priori. This  description  may be in  the form  of a protocol,  study  plan,  report,  or Standard 
179  Operating Procedure (SOP). 
180  2.2.2 Partial Validation     
181  Modifications  to a fully  validated  analytical  method may be evaluated by partial validation. 
182  Partial validation  can range from as little  as one accuracy and precision  determination  to a 
183  nearly full  validation  (Refer to Section 6.1). The items in a partial validation  are determined 
184  according to the extent and nature of the changes made to the method. 
185  2.2.3 Cross Validation    
186  Where data are obtained  from different methods  within  or across studies,  or when data are 
187  obtained within  a study from different laboratories  applying  the same method, comparison of 
188  those data is needed and a cross validation  of the applied analytical  methods should be carried 
189  out (Refer to Section 6.2).   
190  3. CHROMATOGRAPHY 
191  3.1 Reference Standards     
192  During method validation  and the analysis of study samples, a blank biological  matrix is spiked 
193  with the analyte(s) of interest using  solutions  of reference standard(s) to prepare calibration 
194  standards, QCs and stability  QCs. Calibration  standards and QCs should  be prepared from 
195  separate stock solutions.  However, calibration  standards and QCs may be prepared from the 
196  same stock solution provided the accuracy and stability  of the stock solution  have been verified. 
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197  A suitable internal  standard (IS) should  be added to all calibration  standards, QCs and study 
198  samples during sample processing. The absence of an IS should be technically justified.   
199  It is important that the reference standard is well characterised and the quality  (purity, strength, 
200  identity)  of the reference standard and the suitability  of the IS is ensured, as the quality  will 
201  affect the outcome of the analysis and, therefore, the study data. The reference standard used 
202  during validation  and study sample analysis should be obtained from an authentic and traceable 
203  source. The reference standard should  be identical  to the analyte. If this is not possible,  an 
204  established form (e.g., salt or hydrate) of known quality may be used. 
205  Suitable  reference standards include  compendial  standards, commercially  available  standards 
206  or sufficiently  characterised standards prepared in-house  or by an external non-commercial 
207  organisation. A certificate of analysis (CoA) or an equivalent  alternative is required to ensure 
208  quality  and to provide information  on the purity,  storage conditions,  retest/expiration date and 
209  batch number of the reference standard.   
210  A CoA is not required for the IS as long as the suitability  for use is demonstrated, e.g., a lack of 
211  analytical interference is shown for the substance itself or any impurities  thereof.   
212  When  MS  detection  is  used,  the  use  of  the  stable  isotope-labelled  analyte  as the  IS  is 
213  recommended whenever possible. However, it is essential that the labelled standard is of high 
214  isotope purity and that no isotope exchange reaction occurs. The presence of unlabelled  analyte 
215  should  be checked and if  unlabelled  analyte is  detected, the potential  influence  should  be 
216  evaluated during method validation. 
217  Stock and working solutions  can only be prepared from reference standards that are within  the 
218  stability  period  as documented in the CoA (either expiration  date or the retest date in early 
219  development phase). 
220  3.2 Validation 
221  3.2.1 Selectivity         
222  Selectivity  is the ability  of an analytical method to differentiate and measure the analyte in the 
223  presence of potential  interfering substances in the blank biological  matrix.   
224  Selectivity  is evaluated using blank samples (matrix samples processed without addition  of an 
225  analyte  or IS) obtained  from  at least  6 individual  sources/lots  (non-haemolysed  and non-
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226  lipaemic). Use of fewer sources may be acceptable in the case of rare matrices. Selectivity  for 
227  the IS should also be evaluated. 
228  The evaluation  of selectivity  should  demonstrate that no significant  response attributable  to 
229  interfering components is observed at the retention time(s) of the analyte or the IS in the blank 
230  samples. Responses detected and attributable  to interfering  components  should  not be more 
231  than 20% of the analyte response at the LLOQ and not more than 5% of the IS response in the 
232  LLOQ sample for each matrix. 
233  For the investigation  of selectivity  in lipaemic  matrices at least one source of matrix should be 
234  used. To be scientifically  meaningful,  the matrix used for these tests should be representative 
235  as much as possible of the expected study samples. A naturally lipaemic matrix with abnormally 
236  high levels of triglycerides  should be obtained from donors. Although it is recommended to use 
237  lipaemic  matrix from donors, if this is difficult  to obtain,  it is acceptable to spike matrix with 
238  triglycerides  even though it may not be representative of study samples. However, if the drug 
239  impacts lipid  metabolism  or if the intended patient population  is hyperlipidaemic,  the use of 
240  spiked samples is discouraged. This evaluation  is not necessary for preclinical  studies unless 
241  the drug  impacts  lipid  metabolism  or is  administered  in  a particular  animal  strain  that is 
242  hyperlipidaemic.   
243  For the investigation  of selectivity in haemolysed matrices at least one source of matrix should 
244  be used. Haemolysed matrices are obtained by spiking matrix with haemolysed whole blood (at 
245  least 2% V/V) to generate a visibly  detectable haemolysed sample.   
246  3.2.2 Specificity   
247  Specificity  is the ability  of a bioanalytical  method to detect and differentiate the analyte from 
248  other substances, including  its related substances (e.g., substances that are structurally similar 
249  to the analyte,  metabolites,  isomer,  impurities,  degradation  products  formed during  sample 
250  preparation, or concomitant medications that are expected to be used in the treatment of patients 
251  with the intended indication). 
252  If the presence of related substances is anticipated in the biological matrix of interest, the impact 
253  of such substances should be evaluated during  method validation,  or alternatively,  in the pre-
254  dose study  samples.  In the case of  LC-MS based methods,  to assess the impact  of such 
255  substances,  the  evaluation  may  include  comparing  the  molecular  weight  of  a  potential 
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256  interfering  related substance with the analyte and chromatographic  separation of the related 
257  substance from the analyte.    
258  Responses detected and attributable to interfering components should not be more than 20% of 
259  the analyte response at the LLOQ and not more than 5% of the IS response in the LLOQ sample. 
260  The possibility  of back-conversion of a metabolite into the parent analyte during the successive 
261  steps of the analysis  (including  extraction  procedures or in  the MS source) should  also be 
262  evaluated  when  relevant  (i.e.,  potentially  unstable  metabolites  such  as  ester analytes  to 
263  ester/acidic metabolites, unstable N-oxides or glucuronide metabolites, lactone-ring structures). 
264  It  is  acknowledged  that  this  evaluation  will  not  be possible  in  the  early  stages of  drug 
265  development  of a new chemical entity when the metabolism  is not yet evaluated. However, it 
266  is expected that this issue should  be investigated  and partial validation  performed if needed. 
267  The extent of back-conversion, if any, should be established and the impact on the study results 
268  discussed in the Bioanalytical  Report. 
269  3.2.3 Matrix Effect   
270  A matrix effect is defined as an alteration of the analyte response due to interfering  and often 
271  unidentified  component(s) in the sample matrix. During method validation  it is necessary to 
272  evaluate the matrix effect between different independent  sources/lots. 
273  The matrix effect should  be evaluated by analysing  at least 3 replicates of low and high  QCs, 
274  each prepared using matrix from at least 6 different sources/lots. The accuracy should be within 
275  15% of the nominal concentration and the precision (%CV) should not be greater than 15% in 
276  all individual  matrix sources/lots. Use of fewer sources/lots may be acceptable in the case of 
277  rare matrices.   
278  The matrix effect should also be evaluated in relevant patient populations  or special populations 
279  (e.g., hepatically  impaired or renally impaired) when available. An additional  evaluation of the 
280  matrix  effect is recommended using  haemolysed  or lipaemic  matrix  samples during  method 
281  validation  on a case by case basis, especially when these conditions are expected to occur within 
282  the study.   
283  3.2.4 Calibration Curve and Range    
284  The calibration  curve demonstrates the relationship  between the nominal analyte concentration 
285  and the response of the analytical  platform to the analyte. Calibration  standards, prepared by 
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286  spiking  matrix with a known quantity of analyte, span the calibration  range and comprise the 
287  calibration  curve. Calibration  standards should be prepared in the same biological  matrix as the 
288  study samples. The calibration  range is defined by the LLOQ, which is the lowest calibration 
289  standard,  and  the ULOQ,  which  is  the highest  calibration  standard. There should  be one 
290  calibration  curve for each analyte studied during method validation  and for each analytical run. 
291  A calibration  curve should  be generated with  a blank  sample,  a zero sample  (blank sample 
292  spiked with IS), and at least 6 concentration levels of calibration standards, including  the LLOQ 
293  and the ULOQ.  
294  A simple  regression model  that adequately describes the concentration-response  relationship 
295  should be used. The selection of the regression model should be directed by written procedures. 
296  The regression model,  weighting  scheme and transformation  should be determined during  the 
297  method validation.  Blank and zero samples should not be included in the determination  of the 
298  regression equation  for the calibration  curve. Each calibration  standard may be analysed in 
299  replicate,  in which case data from all  acceptable replicates should  be used in the regression 
300  analysis. 
301  The calibration  curve parameters should be reported (slope and intercept in the case of a linear 
302  model). The back-calculated concentrations  of the calibration  standards should  be presented 
303  together  with  the calculated  mean  accuracy values. All  acceptable curves obtained  during 
304  validation,  based on a minimum  of 3 independent runs over several days, should  be reported. 
305  The accuracy of the back-calculated  concentrations  of each calibration  standard  should  be 
306  within 20% of the nominal  concentration at the LLOQ and within 15% at all the other levels. 
307  At least 75% of the calibration standards with a minimum of 6 calibration standard levels should 
308  meet the above criteria.   
309  In the case that replicates are used, the criteria (within  15% or 20% for LLOQ) should also 
310  be fulfilled  for at least 50% of the calibration  standards tested per concentration level. In the 
311  case that a calibration  standard does not comply  with these criteria, this calibration  standard 
312  sample should be rejected, and the calibration curve without this calibration  standard should be 
313  re-evaluated, including  regression analysis. For accuracy and precision runs, if all replicates of 
314  the LLOQ or the ULOQ calibration standard in a run are rejected then the run should be rejected 
315  the possible  source of the failure should be determined and the method revised if necessary. If 
316  the next validation  run also fails, then the method should be revised before restarting validation.   
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317  The calibration  curve should be prepared using freshly spiked calibration  standards in at least 
318  one assessment. Subsequently,  frozen calibration  standards can be used within  their defined 
319  period of stability. 
320  3.2.5 Accuracy and Precision     
321  3.2.5.1 Preparation of Quality Control Samples  
322  The QCs are intended to mimic study samples and should be prepared by spiking  matrix with 
323  a known quantity of analyte, storing  them under the conditions  anticipated for study samples 
324  and analysing  them to assess the validity  of the analytical method. 
325  Calibration  standards and the QCs should be prepared from separate stock solutions  in order to 
326  avoid  biased estimations  which are not related to the analytical  performance of the method. 
327  However, calibration  standards and the QCs may be prepared from the same stock solution, 
328  provided the accuracy and stability  of the stock solution  have been verified. A single source of 
329  blank matrix may be used, which should be free of interference or matrix effects, as described 
330  in Section 3.2.3. 
331  During method validation  the QCs should be prepared at a minimum  of 4 concentration levels 
332  within the calibration curve range: the LLOQ, within three times of the LLOQ (low QC), around 
333  30 - 50% of the calibration curve range (medium QC) and at least 75% of the ULOQ (high QC).  
334  3.2.5.2 Evaluation of Accuracy and Precision  
335  Accuracy and precision  should be determined by analysing  the QCs within  each run (within-
336  run) and in different runs (between-run). Accuracy and precision should be evaluated using the 
337  same runs and data. 
338  Within-run  accuracy and precision should be evaluated by analysing at least 5 replicates at each 
339  QC concentration level in each analytical run. Between-run accuracy and precision should  be 
340  evaluated by analysing  each QC concentration level in at least 3 analytical  runs over at least 
341  two days. To enable the evaluation  of any trends over time within  one run, it is recommended 
342  to demonstrate  accuracy and precision  of  the QCs over at least one of the runs in  a size 
343  equivalent  to a prospective analytical  run of study samples. Reported method validation  data 
344  and the determination  of accuracy and precision should  include all results obtained, including 
345  individual  QCs outside of the acceptance criteria, except those cases where errors are obvious 
346  and documented. Within-run  accuracy and precision data should be reported for each run. If the 
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347  within-run  accuracy or precision criteria are not met in all runs, an overall estimate of within-
348  run accuracy and precision for each QC level should be calculated. Between-run (intermediate) 
349  precision and accuracy should be calculated by combining  the data from all runs.   
350  The calibration curves for these assessments should be prepared using freshly spiked calibration 
351  standards in at least one run. If freshly spiked calibration  standards are not used in the other 
352  runs, stability  of the frozen calibration  standards should be demonstrated. 
353  The overall  accuracy at each concentration  level  should  be within  15%  of the  nominal 
354  concentration,  except at the LLOQ, where it should be within  20%. The precision (%CV) of 
355  the concentrations determined at each level should not exceed 15%, except at the LLOQ, where 
356  it should not exceed 20%. 
357  3.2.6 Carry-over    
358  Carry-over is an alteration of a measured concentration due to residual analyte from a preceding 
359  sample that remains in the analytical instrument.   
360  Carry-over should be assessed and minimised  during method development. During validation 
361  carry-over should be assessed by analysing  blank samples after the calibration  standard at the 
362  ULOQ. Carry-over in the blank samples following  the highest calibration  standard should not 
363  be greater than 20% of the analyte response at the LLOQ and 5% of the response for the IS. If 
364  it appears that carry-over is unavoidable,  study samples should  not be randomised.  Specific 
365  measures should be considered, tested during the validation  and applied during the analysis of 
366  the study samples, so that carry-over does not affect accuracy and precision. This could include 
367  the injection  of blank sample(s) after samples with an expected high concentration, before the 
368  next study sample. 
369  3.2.7 Dilution Integrity  
370  Dilution  integrity is the assessment of the sample dilution  procedure, when required, to confirm 
371  that it does not impact the accuracy and precision of the measured concentration of the analyte. 
372  The same matrix  from the same species used for preparation  of the QCs should  be used for 
373  dilution. 
374  Dilution  QCs should be prepared with analyte concentrations in matrix that are greater than the 
375  ULOQ and then diluted  with blank matrix. At least 5 replicates per dilution  factor should  be 
376  tested in one run to determine if concentrations are accurately and precisely measured within 
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377  the calibration  range. The dilution  ratio(s) applied  during  study  sample analysis  should  be 
378  within  the range of the dilution  ratios evaluated during  validation.  The mean accuracy of the 
379  dilution  QCs should  be within  15% of the nominal  concentration  and the precision  (%CV) 
380  should not exceed 15%. 
381  In the cases of rare matrices, use of a surrogate matrix for dilution  may be acceptable, as long 
382  as it has been demonstrated that this does not affect precision and accuracy.  
383  3.2.8 Stability    
384  Stability  evaluations  should  be carried  out  to ensure that every step taken  during  sample 
385  preparation,  processing and analysis  as well as the storage conditions  used do not affect the 
386  concentration of the analyte.   
387  The storage and analytical  conditions  applied to the stability  tests, such as the sample storage 
388  times and temperatures, sample matrix,  anticoagulant  and container materials,  should  reflect 
389  those used for the study samples. Reference to data published  in the literature is not considered 
390  sufficient. Validation  of storage periods should  be performed on stability  QCs that have been 
391  stored for a time that is equal to or longer than the study sample storage periods. 
392  Stability  of the analyte in the studied  matrix is evaluated using  low and high  concentration 
393  stability  QCs. Aliquots of the low and high stability QCs are analysed at time zero and after the 
394  applied  storage conditions  that are to be evaluated. A minimum  of three stability  QCs should 
395  be prepared and analysed per concentration level/storage condition/timepoint.   
396  The stability  QCs are analysed  against  a calibration  curve,  obtained  from  freshly  spiked 
397  calibration  standards in a run with its corresponding  freshly prepared QCs or QCs for which 
398  stability  has been proven. The mean concentration at each QC level should be within 15% of 
399  the nominal  concentration. If the concentrations  of the study samples are consistently  higher 
400  than the ULOQ of the calibration  range, the concentration of the high stability  QC should  be 
401  adjusted to reflect these higher concentrations. It is recognised that this may not be possible in 
402  nonclinical  studies due to solubility  limitations. 
403  If multiple  analytes are present in the study samples (e.g., studies with a fixed combination,  or 
404  due to a specific drug regimen) the stability  test of an analyte in matrix should be conducted 
405  with the matrix containing  all of the analytes.   
406  The following  stability  tests should be evaluated: 
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407  1)  Stability  of stock and working solutions   
408  The stability  of the stock and working solutions  of the analyte and IS should be determined 
409  under the storage conditions  used during the analysis of study samples by using the lowest 
410  and the highest concentrations of these solutions.  They are assessed using the response of 
411  the  detector.  Stability  of  the  stock  and  working  solutions  should  be  tested  with  an 
412  appropriate  dilution,  taking  into  consideration  the linearity  and measuring  range of the 
413  detector. If the stability  varies with concentration, then the stability  of all concentrations of 
414  the stock and working solutions needs to be assessed. If no isotopic exchange occurs for the 
415  stable isotope-labelled  IS under the same storage conditions  as the analyte for which the 
416  stability is demonstrated, then no additional  stability determinations for the IS are necessary. 
417  If the reference standard expires,  or it is past the retest date, the stability  of the stock 
418  solutions  made previously  with this lot of reference standard are defined by the expiration 
419  or retest date established for the stock solution.  The routine practice of making  stock and 
420  working solutions  from reference standards solely for extending the expiry date for the use 
421  of the reference standard is not acceptable.      
422  2)  Freeze-thaw matrix stability 
423  To assess the impact of repeatedly removing  samples from frozen storage, the stability  of 
424  the analyte should be assessed after multiple  cycles of freezing and thawing. Low and high 
425  stability  QCs should be thawed and analysed according to the same procedures as the study 
426  samples. Stability  QCs should  be kept frozen for at least 12 hours between the thawing 
427  cycles. Stability  QCs for freeze-thaw stability  should  be assessed using  freshly prepared 
428  calibration  standards and QCs or QCs for which stability  has been proven. The number of 
429  freeze-thaw  cycles  validated  should  equal  or  exceed  that  of  the  freeze-thaw  cycles 
430  undergone by the study samples, but a minimum  of three cycles should be conducted. 
431  3)  Bench top (short-term) matrix stability 
432  Bench top matrix  stability  experiments  should  be designed  and conducted  to cover the 
433  laboratory handling  conditions  for the study samples. 
434  Low and high stability QCs should be thawed in the same manner as the study samples and 
435  kept on the bench top at the same temperature and for at least the same duration as the study 
436  samples. 
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437  The total time on the bench top should  be concurrent; it is not acceptable to use additive 
438  exposure to bench top conditions  (i.e., adding up time from each freeze-thaw evaluation is 
439  not acceptable). 
440  4)  Processed sample stability 
441  The stability  of processed samples, including  the time until  completion  of analysis (in the 
442  autosampler/instrument),  should be determined. For example: 
443    Stability  of the processed sample at the storage conditions  to be used during the analysis 
444  of study samples (dry extract or in the injection  phase)   
445    On-instrument/ autosampler stability of the processed sample at injector or autosampler 
446  temperature.   
447  5)  Long-term matrix stability 
448  The long-term stability  of the analyte in matrix stored in the freezer should be established. 
449  Low  and  high  stability  QCs should  be stored  in  the freezer under  the  same storage 
450  conditions  and at least for the same duration as the study samples.   
451  For chemical drugs, it is considered acceptable to extrapolate the stability at one temperature 
452  (e.g., -20C) to lower temperatures (e.g., -70C). 
453  For biological  drugs, it is acceptable to apply a bracketing approach, e.g., in the case that 
454  the stability  has been demonstrated  at -70C  and at -20C,  then it  is not  necessary to 
455  investigate the stability  at temperatures in between those two points at which study samples 
456  will be stored.   
457  In addition,  the following  test should be performed if applicable: 
458  6)  Whole blood stability 
459  Sufficient  attention  should  be paid to the stability  of the analyte in the sampled  matrix 
460  (blood) directly after collection from subjects and prior to preparation for storage to ensure 
461  that the concentrations obtained by the analytical method reflect the concentrations of the 
462  analyte in the subjects blood at the time of sample collection.   
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463  If the matrix  used is plasma  or serum,  the stability  of the analyte  in  blood  should  be 
464  evaluated  during  method  development  (e.g., using  an exploratory  method  in  blood)  or 
465  during method validation.  The results should be provided in the Validation  Report.      
466  3.2.9 Reinjection Reproducibility     
467  Reproducibility  of the method is assessed by replicate measurements of the QCs and is usually 
468  included  in the assessment of precision and accuracy. However, if samples could be reinjected 
469  (e.g., in  the case of  instrument  interruptions  or other  reasons such  as equipment  failure), 
470  reinjection  reproducibility  should  be  evaluated  and  included  in  the  Validation  Report  or 
471  provided in the Bioanalytical  Report of the study where it was conducted. 
472  3.3 Study Sample Analysis     
473  The analysis of study samples can be carried out after validation  has been completed, however, 
474  it  is  understood  that  some parameters may be completed  at a later stage (e.g., long-term 
475  stability).  By the time the data are submitted to a regulatory authority, the bioanalytical  method 
476  validation  should  have been completed.  The study samples,  QCs and calibration  standards 
477  should be processed in accordance with the validated analytical method. If system suitability  is 
478  assessed, a predefined specific study plan, protocol or SOP should be used. System suitability, 
479  including  apparatus conditioning  and instrument  performance,  should  be determined  using 
480  samples that are independent of the calibration  standards and QCs for the run. Subject samples 
481  should  not be used for system suitability.  The IS responses of the study samples should  be 
482  monitored  to  determine  whether  there  is  systemic  IS  variability.  Refer  to  Table  1  for 
483  expectations regarding documentation. 
484  3.3.1 Analytical Run     
485  An analytical  run consists of a blank  sample (processed matrix sample without  analyte and 
486  without IS), a zero sample (processed matrix with IS), calibration  standards at a minimum  of 6 
487  concentration levels, at least 3 levels of QCs (low, medium and high) in duplicate (or at least 
488  5% of the number of study samples, whichever is higher) and the study samples to be analysed. 
489  The QCs should be divided  over the run in such a way that the accuracy and precision  of the 
490  whole run is ensured. Study samples should always be bracketed by QCs.  
491  The calibration  standards and QCs should  be spiked independently  using separately prepared 
492  stock solutions,  unless the accuracy and stability  of the stock solutions  have been verified. All 
493  samples (calibration  standards, QCs and study samples) should be processed and extracted as 
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494  one single batch of samples in the order in which they are intended to be analysed. A single 
495  batch is comprised of study samples and QCs which are handled during a fixed period of time 
496  and by the same group  of analysts with  the same reagents under homogeneous  conditions. 
497  Analysing  samples that were processed as several separate batches in a single analytical run is 
498  discouraged.  If such an approach cannot be avoided,  for instance due to bench top stability 
499  limitations,  each batch of samples should include low, medium and high  QCs. 
500  Acceptance criteria should  be pre-established  in an SOP or in the study plan and should  be 
501  defined  for the whole  analytical  run and the separate batches in  the run,  if applicable.  For 
502  comparative BA/BE studies it is advisable to analyse all samples of one subject together in one 
503  analytical run to reduce variability.   
504  The impact of any carry-over that occurs during study sample analysis should be assessed and 
505  reported  (Refer to  Section  3.2.6).  If  carry-over  is  detected  its  impact  on  the  measured 
506  concentrations  should  be mitigated  (e.g., non-randomisation  of study samples,  injection  of 
507  blank samples after samples with an expected high concentration) or the validity  of the reported 
508  concentrations should be justified  in the Bioanalytical  Report.   
509  3.3.2 Acceptance Criteria for an Analytical Run 
510  Criteria for the acceptance or rejection of an analytical run should be defined in the protocol, in 
511  the study plan or in an SOP. In the case that a run contains multiple  batches, acceptance criteria 
512  should  be applied to the whole run and to the individual  batches. It is possible  for the run to 
513  meet acceptance criteria, even if a batch within that run is rejected for failing  to meet the batch 
514  acceptance criteria. 
515  The back-calculated concentrations of the calibration  standards should  be within  15% of the 
516  nominal  value, except for the LLOQ for which it should be within 20%. At least 75% of the 
517  calibration  standard concentrations, with a minimum  of six concentration levels,  should fulfil 
518  these criteria. If more than 6 calibration  standard levels are used and one of the calibration 
519  standards  does not  meet  the criteria,  this  calibration  standard  should  be rejected  and  the 
520  calibration  curve without this calibration standard should be re-evaluated and a new regression 
521  analysis performed. 
522  If the rejected calibration  standard is the LLOQ, the new lower limit  for this analytical  run is 
523  the next lowest acceptable calibration  standard of the calibration  curve. This new lower limit 
524  calibration  standard  will  retain  its  original  acceptance criteria  (i.e.,  15%).  If the  highest 
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525  calibration  standard is rejected, the ULOQ for this analytical run is the next acceptable highest 
526  calibration  standard of the calibration  curve. The revised calibration range should cover at least 
527  3 QC concentration levels (low, medium and high). Study samples outside of the revised range 
528  should be reanalysed. If replicate calibration  standards are used and only one of the LLOQ or 
529  ULOQ standards fails, the calibration  range is unchanged.   
530  At least 2/3 of the total QCs and at least 50% at each concentration level should be within 15% 
531  of the nominal  values. If these criteria are not fulfilled  the analytical run should be rejected. A 
532  new analytical batch needs to be prepared for all study samples within the failed analytical run 
533  for subsequent analysis. In the cases where the failure is due to an assignable technical cause, 
534  samples may be reinjected. 
535  Analytical runs containing  samples that are diluted and reanalysed should include dilution  QCs 
536  to verify the accuracy and precision of the dilution  method during  study sample analysis. The 
537  concentration of the dilution  QCs should exceed that of the study samples being diluted  (or of 
538  the ULOQ) and they should be diluted using the same dilution  factor. The within-run acceptance 
539  criteria of the dilution  QC(s) will  only affect the acceptance of the diluted  study samples and 
540  not the outcome of the analytical run.   
541  When several analytes are assayed simultaneously,  there should  be one calibration  curve for 
542  each analyte studied. If an analytical run is acceptable for one analyte but has to be rejected for 
543  another analyte, the data for the accepted analyte should  be used. The determination  of the 
544  rejected analyte requires a reextracted analytical batch and analysis. 
545  The  back-calculated  concentrations  of  the  calibration  standards  and  QCs  of  passed  and     
546  accepted runs should be reported. The overall (between-run) accuracy and precision of the QCs 
547  of all  accepted runs  should  be calculated  at each concentration  level  and reported  in  the 
548  analytical report (Refer to Section 8 Documentation and Table 1). If the overall mean accuracy 
549  or precision  fails the 15% criterion,  an investigation  to determine the cause of the deviation 
550  should be conducted. In the case of comparative BA/BE studies it may result in the rejection of 
551  the data. 
552  3.3.3 Calibration Range 
553  If a narrow range of analyte concentrations of the study samples is known or anticipated before 
554  the start of study sample analysis,  it is recommended  to either narrow the calibration  curve 
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555  range, adapt the concentrations of the QCs, or add new QCs at different concentration levels as 
556  appropriate, to adequately reflect the concentrations of the study samples. 
557  At the intended therapeutic dose(s), if an unanticipated  clustering of study samples at one end 
558  of the calibration  curve is encountered after the start of sample analysis, the analysis should be 
559  stopped and either the standard calibration range narrowed (i.e., partial validation),  existing QC 
560  concentrations revised, or QCs at additional  concentrations added to the original  curve within 
561  the observed range before continuing  with study sample analysis. It is not necessary to reanalyse 
562  samples analysed before optimising  the calibration  curve range or QC concentrations. 
563  The same applies if a large number of the analyte concentrations of the study samples are above 
564  the ULOQ. The calibration  curve range should  be changed, if possible,  and QC(s) added or 
565  their concentrations modified.  If it is not possible to change the calibration  curve range or the 
566  number of samples with a concentration above the ULOQ is not large, samples should be diluted 
567  according to the validated dilution  method. 
568  At least 2 QC levels should fall within the range of concentrations measured in study samples. 
569  If the calibration  curve range is changed, the bioanalytical  method should be revalidated (partial 
570  validation)  to verify the response function and to ensure accuracy and precision. 
571  3.3.4 Reanalysis of Study Samples   
572  Possible  reasons for reanalysis  of study samples,  the number of replicates and the decision 
573  criteria to select the value to be reported should  be predefined in the protocol,  study plan or 
574  SOP, before the actual start of the analysis of the study samples. 
575  The number of samples (and percentage of total number of samples) that have been reanalysed 
576  should be reported and discussed in the Bioanalytical  Report.   
577  Some examples of reasons for study sample reanalysis are: 
578    Rejection of an analytical run because the run failed the acceptance criteria with regard to 
579  accuracy of the calibration  standards and/or the precision and accuracy of the QCs 
580    IS response significantly  different from the response for the calibration  standards and QCs 
581  (as pre-defined in an SOP) 
582    The concentration obtained is above the ULOQ 
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583    The concentration observed is below the revised LLOQ in runs where the lowest calibration 
584  standard has been rejected from a calibration  curve, resulting in a higher LLOQ compared 
585  with other runs 
586    Improper sample injection  or malfunction  of equipment 
587    The diluted study sample is below the LLOQ 
588    Identification  of quantifiable  analyte levels in pre-dose samples, control or placebo samples 
589    Poor chromatography (as pre-defined in an SOP) 
590  For comparative BA/BE studies, reanalysis of study samples for a PK reason (e.g., a sample 
591  concentration does not fit with the expected profile) is not acceptable, as it may bias the study 
592  result. 
593  Any reanalysed samples should be identified  in the Bioanalytical  Report and the initial  value, 
594  the reason for reanalysis, the values obtained in the reanalyses, the final accepted value and a 
595  justification  for the acceptance should be provided. Further, a summary table of the total number 
596  of samples that have been reanalysed for each reason should  be provided. In cases where the 
597  first analysis  yields  a non-reportable  result,  a single  reanalysis  is considered sufficient  (e.g., 
598  concentration above the ULOQ or equipment  malfunction).  In cases where the value needs to 
599  be confirmed (e.g., pre-dose sample with measurable concentrations) replicate determinations 
600  are required if sample volume allows.   
601  The  safety  of  trial  subjects  should  take  precedence over  any  other  aspect  of  the  trial. 
602  Consequently, there may be other circumstances when it is necessary to reanalyse specific study 
603  samples for the purpose of an investigation.   
604  3.3.5 Reinjection of Study Samples  
605  Reinjection  of processed samples can be made in the case of equipment  failure if reinjection 
606  reproducibility  has been demonstrated during validation  or provided in the Bioanalytical  Report 
607  where it  was conducted.  Reinjection  of  a full  analytical  run  or  of  individual  calibration 
608  standards or QCs simply because the calibration standards or QCs failed, without any identified 
609  analytical cause, is not acceptable. 
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610  3.3.6 Integration of Chromatograms    
611  Chromatogram  integration  and reintegration  should be described in a study plan,  protocol or 
612  SOP. Any  deviation  from  the  procedures  described  a priori should  be  discussed  in  the 
613  Bioanalytical  Report. The list  of chromatograms  that required  reintegration,  including  any 
614  manual integrations,  and the reasons for reintegration  should be included  in the Bioanalytical 
615  Report. Original  and reintegrated  chromatograms  and  initial  and  repeat integration  results 
616  should be kept for future reference and submitted  in the Bioanalytical  Report for comparative 
617  BA/BE studies. 
618  4. LIGAND BINDING ASSAYS   
619  4.1 Key Reagents 
620  4.1.1 Reference Standard   
621  The reference standard should be well characterised and documented (e.g., CoA and origin). A 
622  biological  drug has a highly  complex  structure and its reactivity  with  binding  reagents for 
623  bioanalysis  may be influenced by a change in the manufacturing process of the drug substance. 
624  It  is  recommended  that  the  manufacturing  batch  of  the  reference standard  used  for  the 
625  preparation of calibration  standards and QCs is derived from the same batch of drug substance 
626  as that used for dosing in the nonclinical  and clinical studies whenever possible. If the reference 
627  standard batch used for bioanalysis  is changed, bioanalytical  evaluation  should be carried out 
628  prior  to  use  to  ensure that  the  performance  characteristics  of the  method  are within  the 
629  acceptance criteria. 
630  4.1.2 Critical Reagents    
631  Critical  reagents, including  binding  reagents (e.g., binding  proteins,  aptamers, antibodies  or 
632  conjugated  antibodies)  and those containing  enzymatic  moieties,  have direct impact  on the 
633  results of the assay and, therefore, their quality  should  be assured. Critical  reagents bind  the 
634  analyte  and,  upon  interaction,  lead  to  an  instrument  signal  corresponding  to  the  analyte 
635  concentration. The critical reagents should be identified  and defined in the assay method.   
636  Reliable  procurement  of  critical  reagents,  whether  manufactured  in-house  or  purchased 
637  commercially,  should be considered early in method development. The data sheet for the critical 
638  reagent should include  at a minimum  identity,  source, batch/lot number, purity (if applicable), 
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639  concentration  (if applicable)  and stability/storage  conditions  (Refer to Table  1). Additional 
640  characteristics may be warranted.  
641  A critical reagent lifecycle management procedure is necessary to ensure consistency between 
642  the original  and new batches of critical  reagents. Reagent performance should  be evaluated 
643  using  the bioanalytical  assay. Minor  changes to critical  reagents would  not be expected to 
644  influence  the  assay  performance,  whereas major  changes  may  significantly  impact  the 
645  performance. If the change is minor  (e.g., the source of one reagent is changed),  a single 
646  comparative accuracy and precision assessment is sufficient for characterisation. If the change 
647  is major,  then additional  validation  experiments are necessary. Ideally,  assessment of changes 
648  will compare the assay with the new reagents to the assay with the old reagents directly. Major 
649  changes include,  but are not limited  to, change in production  method of antibodies,  additional 
650  blood  collection  from animals  for polyclonal  antibodies  and new clones or new supplier  for 
651  monoclonal  antibody production.   
652  Retest dates and validation  parameters should be documented in order to support the extension 
653  or replacement of the critical reagent. Stability  testing of the reagents should be based upon the 
654  performance in the bioanalytical  assay and be based upon general guidance for reagent storage 
655  conditions  and can be extended beyond  the expiry date from the supplier.  The performance 
656  parameters should be documented in order to support the extension or replacement of the critical 
657  reagent.  
658  4.2 Validation    
659  When using LBA, study samples can be analysed using an assay format of 1 or more well(s) 
660  per sample. The assay format should be specified in the protocol, study plan or SOP. If method 
661  development and assay validation  are performed using 1 or more well(s) per sample, then study 
662  sample analysis should also be performed using 1 or more well(s) per sample, respectively. If 
663  multiple  wells  per sample  are used,  the reportable  sample  concentration  value  should  be 
664  determined  either by calculating  the mean of the responses from the replicate wells  or by 
665  averaging  the  concentrations  calculated  from  each response.  Data  evaluation  should  be 
666  performed on reportable concentration values.   
667  4.2.1 Specificity    
668  Specificity is evaluated by spiking  blank matrix samples with related molecules at the maximal 
669  concentration(s) of the structurally related molecule anticipated in study samples.   
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670  The accuracy of the target analyte at the LLOQ and at the ULOQ should be investigated in the 
671  presence of related molecules at the maximal concentration(s) anticipated in study samples. The 
672  response of blank  samples spiked  with  related molecules  should  be below the LLOQ. The 
673  accuracy of the target analyte in presence of related molecules should  be within  25% of the 
674  nominal  values.   
675  In the event of non-specificity,  the impact  on the method  should  be evaluated  by  spiking 
676  increasing concentrations of interfering molecules in blank matrix and measuring the accuracy 
677  of the  target analyte  at the  LLOQ and  ULOQ. It is  essential  to determine  the  minimum 
678  concentration of the related molecule where interference occurs. Appropriate mitigation  during 
679  sample analysis  should  be employed,  e.g., it may be necessary to adjust  the LLOQ/ULOQ 
680  accordingly  or consider a new method. 
681  During  method  development  and  early  assay  validation,  these  related  molecules  are 
682  frequently  not  available.  Additional  evaluation  of specificity  may  be  conducted  after the 
683  original  validation  is completed. 
684  4.2.2 Selectivity    
685  Selectivity  is the ability  of the method to detect and differentiate the analyte of interest in the 
686  presence of other unrelated compounds (non-specific interference) in the sample matrix. The 
687  matrix  can contain  non-specific  matrix  component  such as degrading  enzymes, heterophilic 
688  antibodies or rheumatoid factor which may interfere with the analyte of interest. 
689  Selectivity  should be evaluated at the low end of an assay where problems occur in most cases, 
690  but  it  is  recommended  that selectivity  is  also  evaluated  at higher  analyte  concentrations. 
691  Therefore, selectivity  is evaluated using  blank  samples obtained  from at least 10 individual 
692  sources and by spiking  the individual  blank matrices at the LLOQ and at the high QC level. 
693  The response of the blank samples should be below the LLOQ in at least 80% of the individual 
694  sources.  
695  The accuracy should be within 25% at the LLOQ and within 20% at the high QC level of the 
696  nominal  concentration in at least 80% of the individual  sources evaluated.   
697  Selectivity  should be evaluated in lipaemic  samples and haemolysed samples (Refer to Section 
698  3.2.1). For lipaemic and haemolysed samples, tests can be evaluated once using a single source 
23 
 
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699  of matrix. Selectivity  should  be assessed in samples from relevant patient populations.  In the 
700  case of relevant patient populations  there should be at least five individual  patients.    
701  4.2.3 Calibration Curve and Range  
702  The calibration  curve demonstrates the relationship  between the nominal analyte concentration 
703  and the response of the analytical  platform to the analyte. Calibration  standards, prepared by 
704  spiking  matrix with a known quantity of analyte, span the calibration  range and comprise the 
705  calibration  curve. Calibration  standards should be prepared in the same biological  matrix as the 
706  study samples. The calibration  range is defined by the LLOQ, which is the lowest calibration 
707  standard,  and  the ULOQ,  which  is  the highest  calibration  standard. There should  be one 
708  calibration  curve for each analyte studied during method validation  and for each analytical run. 
709  A calibration  curve should  be generated with  at least 6 concentration  levels  of calibration 
710  standards,  including  LLOQ and ULOQ standards,  plus  a blank  sample. The blank  sample 
711  should not be included in the calculation of calibration curve parameters. Anchor point samples 
712  at concentrations below the LLOQ and above the ULOQ of the calibration  curve may also be 
713  used to  improve  curve fitting.  The relationship  between response and  concentration  for a 
714  calibration  curve is most often fitted by a 4- or 5-parameter logistic  model  if there are data 
715  points near the lower and upper asymptotes, although other models may be used with suitable 
716  justification. 
717  A minimum  of 6 independent runs should be evaluated over several days considering the factors 
718  that may contribute to between-run variability. 
719  The accuracy and precision  of back-calculated  concentrations  of each calibration  standard 
720  should be within 25% of the nominal concentration at the LLOQ and ULOQ, and within 20% 
721  at all  other levels. At least 75% of the calibration  standards excluding  anchor points,  and a 
722  minimum  of 6 concentration levels of calibration  standards, including  the LLOQ and ULOQ, 
723  should meet the above criteria. The anchor points do not require acceptance criteria since they 
724  are beyond the quantifiable  range of the curve. 
725  The calibration  curve should preferably be prepared using freshly spiked calibration  standards. 
726  If freshly spiked calibration standards are not used, the frozen calibration  standards can be used 
727  within their defined period of stability. 
24 
 
  ICH M10 Guideline 
 
 
728  4.2.4 Accuracy and Precision    
729  4.2.4.1 Preparation of Quality Control Samples    
730  The QCs are intended to mimic study samples and should be prepared by spiking  matrix with 
731  a known  quantity  of analyte, stored under the conditions  anticipated  for study samples and 
732  analysed to assess the validity  of the analytical method. 
733  The dilution  series for the preparation of the QCs should be completely  independent from the 
734  dilution  series for the preparation of calibration  standard samples. They may be prepared from 
735  a single  stock provided  that its accuracy has been verified  or is known. The QCs should  be 
736  prepared at a minimum  of 5 concentration levels within the calibration curve range: The analyte 
737  should be spiked at the LLOQ, within three times of the LLOQ (low QC), around the geometric 
738  mean of the calibration  curve range (medium QC), and at least at 75% of the ULOQ (high QC) 
739  and at the ULOQ. 
740  4.2.4.2 Evaluation of Accuracy and Precision   
741  Accuracy and precision  should be determined by analysing  the QCs within  each run (within-
742  run) and in different runs (between-run). Accuracy and precision should be evaluated using the 
743  same runs and data. 
744  Accuracy and precision should be determined by analysing at least 3 replicates per run at each 
745  QC concentration level (LLOQ, low, medium,  high,  ULOQ) in at least 6 runs over 2 or more 
746  days. Reported method validation  data and the determination  of accuracy and precision should 
747  include  all  results obtained,  except those cases where errors are obvious  and documented. 
748  Within-run  accuracy and precision  data should  be reported for each run. If the within-run 
749  accuracy or precision criteria are not met in all runs, an overall estimate of within-run  accuracy 
750  and precision for each QC level should be calculated. Between-run (intermediate) precision and 
751  accuracy should be calculated by combining  the data from all runs. 
752  The overall within-run  and between-run accuracy at each concentration level should be within 
753  20% of the nominal  values, except for the LLOQ and ULOQ, which should be within 25% 
754  of  the  nominal  value.  Within-run  and  between-run  precision  of  the  QC  concentrations 
755  determined  at each level should  not exceed 20%, except at the LLOQ and ULOQ, where it 
756  should not exceed 25%.   
25 
 
  ICH M10 Guideline 
 
 
757  Furthermore,  the total  error (i.e.,  sum of absolute  value of the errors in  accuracy (%) and 
758  precision (%)) should be evaluated. The total error should not exceed 30% (40% at LLOQ and 
759  ULOQ). 
760  4.2.5 Carry-over 
761  Carry-over is generally not an issue for LBA analyses. However, if the assay platform is prone 
762  to carry-over, the potential of carry-over should be investigated  by placing blank samples after 
763  the calibration  standard at the ULOQ. The response of blank  samples  should  be below  the 
764  LLOQ. 
765  4.2.6 Dilution Linearity and Hook Effect   
766  Due to the narrow assay range in many LBAs, study samples may require dilution  in order to 
767  achieve analyte concentrations within  the range of the assay. Dilution  linearity  is assessed to 
768  confirm:  (i) that measured concentrations  are not affected by dilution  within  the calibration 
769  range and (ii)  that sample  concentrations  above the ULOQ of a calibration  curve are not 
770  impacted by hook effect (i.e., a signal suppression caused by high concentrations of the analyte), 
771  whereby yielding  an erroneous result. 
772  The same matrix as that of the study sample should  be used for preparation  of the QCs for 
773  dilution. 
774  Dilution  linearity  should be demonstrated by generating a dilution  QC, i.e., spiking  the matrix 
775  with  an analyte  concentration  above  the ULOQ, analysed  undiluted  (for hook  effect) and 
776  diluting  this sample (to at least 3 different dilution  factors) with blank matrix to a concentration 
777  within the calibration range. For each dilution  factor tested, at least 3 runs should be performed 
778  using the number of replicates that will be used in sample analysis. The absence or presence of 
779  response reduction  (hook  effect) is checked in the dilution  QCs and, if observed, measures 
780  should be taken to eliminate  response reduction during the analysis of study samples. 
781  The  calculated  concentration  for  each  dilution  should  be  within  20%  of  the  nominal 
782  concentration after correction for dilution  and the precision of the final  concentrations across 
783  all the dilutions  should not exceed 20%.   
784  The dilution  factor(s) applied  during  study  sample  analysis  should  be within  the range of 
785  dilution  factors evaluated during validation. 
26 
 
  ICH M10 Guideline 
 
 
786  4.2.7 Stability    
787  Stability  evaluations  should  be carried  out  to ensure that every step taken  during  sample 
788  preparation,  processing and analysis  as well as the storage conditions  used do not affect the 
789  concentration of the analyte.   
790  The storage and analytical  conditions  applied to the stability  tests, such as the sample storage 
791  times and temperatures, sample matrix,  anticoagulant,  and container materials  should  reflect 
792  those used for the study samples. Reference to data published  in the literature is not considered 
793  sufficient. Validation  of storage periods should  be performed on stability  QCs that have been 
794  stored for a time that is equal to or longer than the study sample storage periods. 
795  Stability  of the analyte in the studied  matrix is evaluated using  low and high  concentration 
796  stability  QCs. Aliquots of the low and high stability  QCs are analysed at time zero and after the 
797  applied  storage conditions  that are to be evaluated. A minimum  of three stability  QCs should 
798  be prepared and analysed per concentration level/storage condition/timepoint.   
799  The stability  QCs are analysed  against  a calibration  curve,  obtained  from  freshly  spiked 
800  calibration  standards in a run with its corresponding  freshly prepared QCs or QCs for which 
801  stability  has been proven. While the use of freshly prepared calibration  standards and QCs is 
802  the preferred approach,  it is recognised  that in  some cases, for macromolecules,  it may be 
803  necessary to freeze them overnight.  In such cases, valid  justification  should  be provided  and 
804  freeze-thaw stability  demonstrated. The mean concentration  at each level  should  be within 
805  20% of the nominal  concentration.   
806  Since sample dilution  may be required for many LBA assays due to a narrow calibration  range, 
807  the concentrations  of the study  samples may be consistently  higher  than the ULOQ of the 
808  calibration  curve. If this is the case, the concentration of the stability  QCs should be adjusted, 
809  considering  the applied sample dilution,  to represent the actual sample concentration range. 
810  As mentioned in Section 3.2.8, the investigation  of stability should cover bench top (short-term) 
811  stability  at room temperature or sample preparation temperature and freeze-thaw stability.  In 
812  addition,  long-term stability  should be studied.   
813  For chemical drugs, it is considered acceptable to extrapolate the stability  at one temperature 
814  (e.g., -20C) to lower temperatures (e.g., -70C). 
27 
 
  ICH M10 Guideline 
 
 
815  For biological  drugs, it is acceptable to apply a bracketing approach, e.g., in the case that the 
816  stability  has been demonstrated at -70C and at -20C, then it is not necessary to investigate the 
817  stability  at temperatures in between those two points at which study samples will be stored.   
818  4.3 Study Sample Analysis 
819  The analysis of study samples can be carried out after validation  has been completed however 
820  it  is  understood  that  some parameters may be completed  at a later stage (e.g., long-term 
821  stability).  By the time the data are submitted to a regulatory authority, the bioanalytical  method 
822  validation  should  have been completed.  The study samples,  QCs and calibration  standards 
823  should  be processed in accordance with the validated analytical  method. Refer to Table 1 for 
824  expectations regarding documentation. 
825  4.3.1 Analytical Run 
826  An  analytical  run  consists  of  a blank  sample,  calibration  standards  at  a minimum  of  6 
827  concentration levels, at least 3 levels of QCs (low, medium and high) applied as two sets (or at 
828  least 5% of the number of study samples, whichever is higher) and the study samples to be 
829  analysed. The blank  sample  should  not  be included  in  the calculation  of calibration  curve 
830  parameters. The QCs should be placed in the run in such a way that the accuracy and precision 
831  of the whole run is ensured taking into account that study samples should always be bracketed 
832  by QCs. 
833  Most often microtitre plates are used for LBAs. An analytical run may comprise of one or more 
834  plate(s). Typically,  each plate contains an individual  set of calibration  standards and QCs. If 
835  each plate contains its own calibration  standards and QCs then each plate should be assessed 
836  on its own. However, for some platforms the sample capacity may be limited.  In this case, sets 
837  of calibration  standards may be placed on the first and the last plate, but QCs should be placed 
838  on every single  plate. QCs should  be placed at least at the beginning  (before) and at the end 
839  (after) of the study samples of each plate. The QCs on each plate and each calibration  curve 
840  should  fulfil  the  acceptance  criteria  (Refer  to  Section  4.3.2).  For  the  calculation  of 
841  concentrations, the calibration  standards should be combined to conduct one regression analysis. 
842  If the combined calibration  curve does not pass the acceptance criteria the whole run fails. 
843  4.3.2 Acceptance Criteria for an Analytical Run    
844  Criteria for the acceptance or rejection of an analytical run should be defined in the protocol, in 
845  the study plan or in an SOP. In the case that a run contains multiple  batches, acceptance criteria 
28 
 
  ICH M10 Guideline 
 
 
846  should  be applied to the whole run and to the individual  batches. It is possible  for the run to 
847  meet acceptance criteria, even if a batch within that run is rejected for failing  to meet the batch 
848  acceptance criteria. 
849  The back-calculated concentrations of the calibration  standards should  be within  20% of the 
850  nominal  value at each concentration level, except for the LLOQ and the ULOQ, for which it 
851  should  be within  25%. At least 75% of the calibration  standards,  with  a minimum  of 6 
852  concentration  levels,  should  fulfil  this  criterion.  This requirement  does not apply  to anchor 
853  calibration  standards. If more than 6 calibration  standards are used and one of the calibration 
854  standards does not  meet these criteria,  this  calibration  standard should  be rejected and the 
855  calibration  curve without this calibration standard should be re-evaluated and a new regression 
856  analysis performed. 
857  If the rejected calibration  standard is the LLOQ, the new lower limit  for this analytical  run is 
858  the next lowest acceptable calibration standard of the calibration  curve. If the highest calibration 
859  standard is rejected, the new upper limit  for this analytical  run is the next acceptable highest 
860  calibration standard of the calibration curve. The new lower and upper limit calibration standard 
861  will  retain their original  acceptance criteria (i.e., 20%). The revised calibration  range should 
862  cover all QCs (low, medium  and high). The study samples outside of the revised assay range 
863  should be reanalysed. 
864  Each run should contain at least 3 levels of QCs (low, medium and high). During study sample 
865  analysis, the calibration standards and QCs should mimic the analysis of the study sample with 
866  regard to the number of wells used per study sample. At least 2/3 of the QCs and 50% at each 
867  concentration level should  be within  20% of the nominal  value at each concentration level. 
868  Exceptions to these criteria should be justified  and predefined in the SOP or protocol. 
869  The overall mean accuracy and precision of the QCs of all accepted runs should be calculated 
870  at each concentration  level and reported in the analytical  report. In the case that the overall 
871  mean accuracy and/or precision exceeds 20%, additional  investigations  should be conducted to 
872  determine the cause(s) of this deviation. In the case of comparative BA/BE studies it may result 
873  in the rejection of the data. 
874  4.3.3 Calibration Range  
875  At least 2 QC sample levels should  fall within  the range of concentrations measured in study 
876  samples. At the intended therapeutic dose(s), if an unanticipated clustering of study samples at 
29 
 
  ICH M10 Guideline 
 
 
877  one end of the calibration  curve is encountered after the start of sample analysis,  the analysis 
878  should be stopped and either the standard calibration  range narrowed (i.e., partial validation), 
879  existing  QC concentrations revised, or QCs at additional  concentrations added to the original 
880  curve  within  the  observed  range  before  continuing  with  study  sample  analysis.  It is  not 
881  necessary to reanalyse samples analysed before optimising  the calibration  curve range or QC 
882  concentrations. 
883  4.3.4 Reanalysis of Study Samples 
884  Possible reasons for reanalysis of study samples, the number of reanalyses and the decision 
885  criteria to select the value to be reported should  be predefined in the protocol,  study plan or 
886  SOP, before the actual start of the analysis of the study samples. 
887  The number of samples (and percentage of total number of samples) that have been reanalysed 
888  should be reported and discussed in the Bioanalytical  Report. 
889  Some examples of reasons for study sample reanalysis are: 
890    Rejection of an analytical run because the run failed the acceptance criteria with regard to 
891  accuracy of the calibration  standards and/or the precision and accuracy of the QCs 
892    The concentration obtained is above the ULOQ 
893    The  concentration  obtained  is  below  the LLOQ in  runs  where the  lowest  calibration 
894  standard has been rejected from a calibration  curve, resulting in a higher LLOQ compared 
895  with other runs 
896    Malfunction  of equipment 
897    The diluted sample is below the LLOQ 
898    Identification  of quantifiable  analyte levels in pre-dose samples, control or placebo samples. 
899    When samples are analysed in more than one well and non-reportable  values are obtained 
900  due to one replicate failing  the pre-defined acceptance criteria (e.g., excessive variability 
901  between wells, one replicate being above the ULOQ or below the LLOQ). 
902  For comparative BA/BE studies, reanalysis of study samples for a PK reason (e.g., a sample 
903  concentration does not fit with the expected profile) is not acceptable, as it may bias the study 
904  result. 
30 
 
  ICH M10 Guideline 
 
 
905  The reanalysed samples should be identified  in the Bioanalytical  Report and the initial  value, 
906  the reason for reanalysis, the values obtained in the reanalyses, the final accepted value and a 
907  justification  for the acceptance should be provided. Further, a summary table of the total number 
908  of samples that have been reanalysed due to each reason should be provided. In cases where the 
909  first analysis  yields  a non-reportable  result,  a single  reanalysis  is considered sufficient  (e.g., 
910  concentration  above the ULOQ or excessive variability  between wells). The analysis  of the 
911  samples should be based on the same number of wells per study sample as in the initial  analysis. 
912  In cases where the value  needs  to be confirmed,  (e.g., pre-dose  sample  with  measurable 
913  concentrations) multiple  determinations  are required where sample volume allows.   
914  The  safety  of  trial  subjects  should  take  precedence over  any  other  aspect  of  the  trial. 
915  Consequently, there may be other circumstances when it is necessary to reanalyse specific study 
916  samples for the purpose of an investigation.   
917  5. INCURRED SAMPLE REANALYSIS (ISR)        
918  The performance of study samples may differ from that of the calibration  standards and QCs 
919  used during  method validation,  which are prepared by spiking  blank matrix.  Differences in 
920  protein binding,  back-conversion of known and unknown metabolites,  sample inhomogeneity, 
921  concomitant medications or biological  components unique to the study samples may affect the 
922  accuracy and precision of analysis of the analyte in study samples. 
923  Therefore, ISR is a necessary component  of bioanalytical  method validation.  It is intended to 
924  verify the reliability  of the reported sample analyte concentrations and to critically  support the 
925  precision and accuracy measurements established with spiked QCs. 
926  ISR should be performed at least in the following  situations: 
927    For preclinical  studies, ISR should,  in general, be performed for the main nonclinical  TK 
928  studies once per species. However, ISR in a PK study instead of a TK study might also be 
929  acceptable, as long as the respective study has been conducted as a pivotal  study, used to 
930  make regulatory decisions. 
931    All pivotal comparative BA/BE studies 
932    First clinical  trial in subjects 
933    Pivotal early patient trial(s), once per patient population 
31 
 
  ICH M10 Guideline 
 
 
934    First or pivotal trial in patients with impaired hepatic and/or renal function 
935  ISR is conducted by repeating the analysis of a subset of samples from a given study in separate 
936  (i.e., different to the original)  runs on different days using the same bioanalytical  method. 
937  The extent of ISR depends upon the analyte and the study samples and should be based upon 
938  an in-depth understanding of the analytical method and analyte. However, as a minimum,  if the 
939  total number of study samples is less than 1000, then 10% of the samples should be reanalysed; 
940  if the total number of samples is greater than 1000,  then 10% of the first 1000 samples (100) 
941  plus 5% of the number of samples that exceed 1000 samples should  be assessed. Objective 
942  criteria for choosing the subset of study samples for ISR should be predefined in the protocol, 
943  study plan or an SOP. While the subjects should be picked as randomly  as possible  from the 
944  dosed  study  population,  adequate  coverage of the PK profile  in  its  entirety  is  important. 
945  Therefore,  it  is  recommended  that  the samples  for  ISR be  chosen  around  the  maximum 
946  concentration  (Cmax) and some in  the elimination  phase. Additionally,  the samples chosen 
947  should be representative of the whole study. 
948  Samples should not be pooled, as pooling  may limit  anomalous findings.  ISR samples and QCs 
949  should  be prepared in the same manner as in the original  analysis.  ISR should be performed 
950  within the stability  window of the analyte, but not on the same day as the original  analysis.   
951  The percent difference between the initial  concentration and the concentration measured during 
952  the repeat analysis  should  be calculated  in relation  to their  mean value using  the following 
953  equation:   
repeat value initial value
954  % difference =    100 
mean value
955   
956  For chromatographic  methods, the percent difference should be  20% for at least 2/3 of the 
957  repeats. For LBAs, the percent difference should be  30% for at least 2/3 of the repeats. 
958  If the overall ISR results fail the acceptance criteria, an investigation  should be conducted and 
959  the causes remediated. There should  be an SOP that directs how investigations  are triggered 
960  and conducted. If an investigation  does not identify the cause of the failure, the potential impact 
961  of an ISR failure on study validity  should also be provided  in the Bioanalytical  Report. If ISR 
962  meets the acceptance criteria  yet shows large  or  systemic  differences  between results  for 
32 
 
  ICH M10 Guideline 
 
 
963  multiple  samples, this may indicate analytical issues and it is advisable to investigate this further.
964     
965  Examples of trends that are of concern include: 
966    All samples from one subject fail 
967    All of samples from one run fail 
968  All aspects of ISR evaluations should be documented to allow reconstruction of the study and 
969  any investigations.  Individual  samples that are quite different from the original  value (e.g., > 
970  50%,  flyers) should  not trigger  reanalysis  of the original  sample  and do not need to be 
971  investigated. ISR sample data should not replace the original  study sample data. 
972  6. PARTIAL AND CROSS VALIDATION     
973  6.1 Partial Validation 
974  Partial  validations  evaluate modifications  to  already fully  validated  bioanalytical  methods. 
975  Partial  validation  can  range  from  as  little  as  one  within-run  accuracy  and  precision 
976  determination,  to a nearly full  validation.  If stability  is established  at one facility  it does not 
977  necessarily need to be repeated at another facility.   
978  For chromatographic  methods, typical bioanalytical  method modifications  or changes that fall 
979  into this category include,  but are not limited  to, the following  situations: 
980    Analytical  site change using  same method  (i.e., bioanalytical  method  transfers between 
981  laboratories) 
982    A change in analytical methodology  (e.g., change in detection systems, platform) 
983    A change in sample processing procedures   
984    A change in sample volume (e.g., the smaller volume of paediatric samples)   
985    Changes to the calibration  concentration range 
986    A change in anticoagulant  (but not changes in the counter-ion)  in biological  fluids  (e.g., 
987  heparin to EDTA) 
33 
 
  ICH M10 Guideline 
 
 
988    Change from one matrix within a species to another (e.g., switching from human plasma to 
989  serum or cerebrospinal fluid)  or changes to the species within  the matrix (e.g., switching 
990  from rat plasma to mouse plasma)   
991    A change in storage conditions 
992  For LBAs, typical bioanalytical  method modifications  or changes that fall  into  this category 
993  include,  but are not limited  to, the following  situations: 
994    Changes in LBA critical reagents (e.g., lot-to-lot  changes) 
995    Changes in MRD 
996    A change in storage conditions 
997    Changes to the calibration  concentration range 
998    A change in analytical methodology  (e.g., change in detection systems, platform) 
999    Analytical site change using same method (i.e., bioanalytical  method transfers between 
1000  laboratories) 
1001    A change in sample preparation   
1002  Partial validations  are acceptable if the parameters tested meet the full  validation  criteria. If 
1003  these criteria are not satisfied, additional  investigation  and validation  is warranted. 
1004  6.2 Cross Validation 
1005  Cross validation  is required to compare data under the following  situations: 
1006    Data are obtained from different fully validated methods within a study. 
1007    Data are obtained from different fully validated methods across studies that are going to be 
1008  combined  or  compared  to  support  special  dosing  regimens,  or  regulatory  decisions 
1009  regarding safety, efficacy and labelling. 
1010    Data are obtained  within  a study from different laboratories  with the same bioanalytical 
1011  method.   
1012  Cross validation  is not generally required to compare data obtained across studies from different 
1013  laboratories using the same validated method at each site. 
34 
 
  ICH M10 Guideline 
 
 
1014  Cross validation  should be performed in advance of study samples being analysed, if possible. 
1015  Cross validation  should be assessed by measuring the same set of QCs (low, medium and high) 
1016  in triplicate  and study  samples that span the study sample concentration  range (if available 
1017  n30) with both assays or in both laboratories.   
1018  Bias can be assessed by Bland-Altman  plots or Deming regression. Other methods appropriate 
1019  for assessing agreement between two assays (e.g., concordance correlation coefficient) may be 
1020  used too. Alternatively,  the concentration vs. time curves for incurred samples could be plotted 
1021  for samples  analysed  by each method  to assess bias.  If disproportionate  bias  is  observed 
1022  between methods, the impact on the clinical data interpretation  should be assessed. 
1023  The use of multiple  bioanalytical  methods in the conduct of one comparative BA/BE study is 
1024  strongly discouraged. 
1025  7. ADDITIONAL CONSIDERATIONS    
1026  7.1 Analytes that are also Endogenous Compounds     
1027  For analytes that are also endogenous  compounds,  the accuracy of the measurement of the 
1028  analytes poses a challenge when the assay cannot distinguish  between the therapeutic agent and 
1029  the endogenous counterpart. 
1030  The endogenous levels may vary because of age, gender, diurnal variations,  illness or as a side 
1031  effect of drug treatment. If available,  biological  matrix with an adequate signal-to-noise  ratio 
1032  (i.e., endogenous level sufficiently  low for the desired LLOQ, e.g., <20% of the LLOQ) should 
1033  be used as blank matrix to prepare calibration  standards and QCs since the biological  matrix 
1034  used to prepare calibration  standards and QCs should be the same as the study samples (i.e., 
1035  authentic biological  matrix) and should be free of matrix effect and endogenous analyte at the 
1036  level that causes interference. 
1037  In those cases where matrices without  interference are not available,  there are four possible 
1038  approaches to calculate the concentration of the endogenous analyte in calibration  standards, 
1039  QCs and, consequently,  study samples:  1) the standard addition  approach, 2) the background 
1040  subtraction approach, 3) the surrogate matrix (neat, artificial or stripped matrices) approach and 
1041  4) the surrogate analyte approach. 
1042  1)  Standard Addition  Approach: 
35 
 
  ICH M10 Guideline 
 
 
1043  Every study sample is divided  into aliquots  of equal volume. All aliquots,  but one, 
1044  are separately spiked with known and varying amounts of the analyte standards to 
1045  construct a calibration curve for every study sample. The study sample concentration 
1046  is  then  determined  as the  negative  x-intercept  of the standard  calibration  curve 
1047  prepared in that particular study sample. 
1048  2)  Background Subtraction Approach: 
1049  The endogenous  background  concentrations  of analytes in  a pooled/representative 
1050  matrix are subtracted from the concentrations  of the added standards, subsequently 
1051  the subtracted concentrations are used to construct the calibration  curve. 
1052  3)  Surrogate Matrix Approach: 
1053  The matrix  of the study  samples  is substituted  by a surrogate matrix.  Surrogate 
1054  matrices can vary widely in complexity  from simple buffers or artificial matrices that 
1055  try to mimic the authentic one, to stripped matrices. 
1056  4)  Surrogate Analyte Approach: 
1057  Stable-isotope  labelled  analytes  are used  as surrogate  standards to  construct  the 
1058  calibration  curves for the quantification  of endogenous analytes. In this method it is 
1059  assumed that the physicochemical  properties of the authentic and surrogates analytes 
1060  are the same with the exception of molecular weight. However, isotope standards may 
1061  differ  in  retention  time  and  MS sensitivity,  therefore,  before application  of this 
1062  approach,  the  ratio  of the labelled  to  unlabelled  analyte  MS  responses (i.e.,  the 
1063  response factor) should be close to unity and constant over the entire calibration range. 
1064  If  the  response  factor  does  not  comply  with  these  requirements,  it  should  be 
1065  incorporated into the regression equation of the calibration  curve. 
1066  Validation  of an analytical  method for an analyte that is also an endogenous  compound  will 
1067  require the following  considerations. 
1068  7.1.1 Quality Control Samples 
1069  The endogenous concentrations of the analyte in the biological  matrix should be evaluated prior 
1070  to QC preparation (e.g., by replicate analysis). The blank matrices with the minimum  level of 
1071  the endogenous analyte should be used. The concentrations of the QCs should account for the 
36 
 
  ICH M10 Guideline 
 
 
1072  endogenous concentrations in the biological  matrix (i.e., additive) and be representative of the 
1073  expected study concentrations. 
1074  The QCs used for validation  should be aliquots of the authentic biological  matrix unspiked and 
1075  spiked  with known  amounts of the authentic  analyte. In spiked  samples, the added amount 
1076  should be enough to provide concentrations that are statistically  different from the endogenous 
1077  concentration. 
1078  7.1.2 Calibration Standards 
1079  In the Surrogate Matrix and Surrogate Analyte Approaches, these surrogates should  be used 
1080  only for the preparation of the calibration  standards. 
1081  In the Standard Addition  and Background Subtraction Approaches the same biological  matrix 
1082  and analyte as the study samples is used to prepare the calibration  standards. However, when 
1083  the background  concentrations  are lowered by dilution  of the blank  matrices before spiking 
1084  with the standards (e.g., if a lower LLOQ is required in the Background Subtraction Approach) 
1085  the composition  of the matrices in the study samples and the calibration  standards is different, 
1086  which may cause different recoveries and matrix effects. 
1087  7.1.3 Selectivity, Recovery and Matrix Effects 
1088  The assessment of selectivity  is complicated  by the absence of interference-free matrix. For 
1089  chromatography,  peak purity should be investigated  as part of method validation  by analysing 
1090  matrices obtained from several donors using a discriminative  detection system (e.g., MS/MS). 
1091  Other approaches, if justified  by scientific principles,  may also be considered. 
1092  For the Standard Addition  and Background  Subtraction Approaches, as the same biological 
1093  matrix and analyte are used for study samples and calibration standards, the same recovery and 
1094  matrix effect occurs in the study samples and the calibration standards. For the Surrogate Matrix 
1095  and Surrogate Analyte Approaches, the matrix  effect and the extraction recovery may differ 
1096  between calibration  standards and study samples. 
1097    If the Surrogate Matrix Approach is used, demonstration  of similar  matrix effect and 
1098  extraction recovery in both the surrogate and original  matrix is required. This should be 
1099  investigated  in an experiment using QCs spiked with analyte in the matrix against the 
1100  surrogate calibration  curve and should be within  15% for chromatographic assays and 
1101  within 20% for LBA assays.  
37 
 
  ICH M10 Guideline 
 
 
1102    If the Surrogate Analyte Approach is used, demonstration  of similarity  in matrix effect 
1103  and recovery between surrogate and authentic endogenous  analytes is required. This 
1104  should be investigated  in an experiment within 15% for chromatographic  assays and 
1105  within 20% for LBA assays.  
1106  Since the composition  of the biological  matrix might affect method performance, it is necessary 
1107  to investigate matrices from different donors, except in the Standard Addition Approach, where 
1108  each sample is analysed with its own calibration  curve. 
1109  7.1.4 Parallelism 
1110  Parallelism should be evaluated in the Surrogate Matrix and Surrogate Analyte Approaches by 
1111  means of the Standard Addition  approach, spike recovery or dilutional  linearity. 
1112  7.1.5 Accuracy and Precision 
1113  In case of using a surrogate matrix or analyte, the assessment of accuracy and precision should 
1114  be performed by analysing  the QCs against the surrogate calibration  curve. In certain cases, 
1115  dilution  of the QCs with  surrogate matrix  may be necessary. These experiments  should  be 
1116  repeated with authentic biological  matrices from different donors to address variability  due to 
1117  the  matrix.  Analysis  of  the  unspiked  QCs  will  give  the  mean  endogenous  background 
1118  concentration and only precision and no accuracy can be determined for this QCs. 
1119  The concentration  of the endogenous  substance in the blank sample may be determined and 
1120  subtracted  from  the  total  concentrations  observed  in  the  spiked  samples.  Accuracy  is 
1121  recommended to be calculated using the following  formula:  
(Measured concentration of spiked sampleendogenous concentration )
1122  Accuracy (%)=100   
Nominal concentration 
1123   
1124  7.1.6 Stability 
1125  In  order  to  mimic  study  samples  as  much  as  possible,  stability  experiments  should  be 
1126  investigated with the authentic analyte in the authentic biological  matrix and with unspiked and 
1127  spiked samples. However, if a surrogate matrix is used for calibration standards, stability should 
1128  also be demonstrated for the analyte in the surrogate matrix, as this could differ from stability 
1129  in the authentic biological  matrix. 
38 
 
  ICH M10 Guideline 
 
 
1130  7.2 Parallelism     
1131  Parallelism  is defined  as a parallel  relationship  between the calibration  curve and serially 
1132  diluted  study samples to detect any influence  of dilution  on analyte measurement. Although 
1133  lack of parallelism  is a rare occurrence for PK assays, parallelism  of LBA should be evaluated 
1134  on a case-by-case basis, e.g., where interference caused by a matrix component (e.g., presence 
1135  of  endogenous  binding  protein)  is  suspected  during  study  sample  analysis.  Parallelism 
1136  investigation  or the justification  for its absence should be included in the Bioanalytical  Report. 
1137  As  parallelism  assessments  are rarely  possible  during  method  development  and  method 
1138  validation  due to the unavailability  of study samples and parallelism  is strictly  linked  to the 
1139  study samples (i.e., an assay may have perfectly suitable parallelism  for a certain population  of 
1140  samples, yet lack it for another population),  these experiments should be conducted during the 
1141  analysis of the study samples. A high concentration  study sample (preferably close to Cmax) 
1142  should  be diluted  to at least three concentrations  with blank  matrix. The precision  between 
1143  samples in a dilution  series should not exceed 30%. However, when applying the 30% criterion, 
1144  data should  be carefully monitored  as results that pass this criterion may still  reveal trends of 
1145  non-parallelism.  In the case that the sample does not dilute  linearly  (i.e., in  a non-parallel 
1146  manner), a procedure for reporting a result should be defined a priori. 
1147  7.3 Recovery  
1148  For methods  that employ  sample extraction,  the recovery (extraction  efficiency)  should  be 
1149  evaluated. Recovery is reported as a percentage of the known  amount of an analyte carried 
1150  through the sample extraction and processing steps of the method. Recovery is determined by 
1151  comparing  the analyte response in  a biological  sample that is spiked  with  the analyte and 
1152  processed, with the response in a biological  blank sample that is processed and then spiked with 
1153  the analyte. Recovery of the analyte does not need to be 100%, but the extent of recovery of an 
1154  analyte and of the IS (if used) should be consistent. Recovery experiments are recommended to 
1155  be  performed  by  comparing  the  analytical  results  for  extracted  samples  at  multiple 
1156  concentrations, typically  three concentrations (low, medium and high). 
1157  7.4 Minimum Required Dilution     
1158  MRD is a dilution  factor employed  in samples that are diluted  with buffer solution  to reduce 
1159  the background signal or matrix interference on the analysis using LBA. The MRD should be 
1160  identical  for  all  samples  including  calibration  standards  and  the  QCs  and  it  should  be 
1161  determined during method development. If MRD is changed after establishment of the method, 
39 
 
  ICH M10 Guideline 
 
 
1162  partial validation  is necessary. MRD should be defined in the Validation  Report of the analytical 
1163  method. 
1164  7.5 Commercial and Diagnostic Kits   
1165  Commercial or diagnostic kits (referred to as kits) are sometimes co-developed with new drugs 
1166  or therapeutic biological  products for point-of-care patient diagnosis. The recommendations in 
1167  this section of the guideline  do not apply to the development of kits that are intended for point-
1168  of-care patient  diagnosis  (e.g., companion  or complimentary  diagnostic  kits).  Refer to  the 
1169  appropriate  guideline  documents  regarding  regulatory  expectations  for the development  of 
1170  these kits.   
1171  If an applicant  repurposes a kit (instead of developing  a new assay) or utilises  research use 
1172  only kits to measure chemical or biological  drug concentrations during the development  of a 
1173  novel drug, the applicant should assess the kit validation  to ensure that it conforms to the drug 
1174  development standards described in this guideline. 
1175  Validation  considerations  for kit assays include, but are not limited  to, the following: 
1176    If the reference standard in the kit differs from that of the study samples, testing should 
1177  evaluate differences in assay performance of the kit reagents. The specificity,  accuracy, 
1178  precision and stability  of the assay should  be demonstrated under actual conditions  of use 
1179  in  the  facility  conducting  the  sample  analysis.  Modifications  from  kit  processing 
1180  instructions  should be completely  validated.     
1181    Kits that use sparse calibration  standards (e.g., one- or two-point calibration  curves) should 
1182  include in-house validation  experiments to establish the calibration  curve with a sufficient 
1183  number of standards across the calibration range. 
1184    Actual QC concentrations should be known. Concentrations of QCs expressed as ranges are 
1185  not sufficient for quantitative  applications.  In such cases QCs with known concentrations 
1186  should be prepared and used, independent of the kit-supplied  QCs. 
1187    Calibration  standards and QCs should be prepared in the same matrix as the study samples. 
1188  Kits with  calibration  standards and QCs prepared in  a matrix  different  from the  study 
1189  samples should be justified  and appropriate experiments should be performed.   
40 
 
  ICH M10 Guideline 
 
 
1190    If multiple  kit lots are used within a study, lot-to-lot variability  and comparability  should be 
1191  addressed for any critical reagents included in the kits.     
1192    If a kit using multiple  assay plates is employed,  sufficient replicate QCs should be used on 
1193  each plate to monitor  the accuracy of the assay. Acceptance criteria should be established 
1194  for the individual  plates and for the overall analytical run.      
1195  7.6 New or Alternative Technologies    
1196  When a new or alternative technology  is used as the sole bioanalytical  technology  from the 
1197  onset of drug development,  cross validation  with an existing technology is not required.   
1198  The use of two different bioanalytical  technologies  for the development of a drug may generate 
1199  data for the same product that could be difficult  to interpret. This outcome can occur when one 
1200  platform generates drug concentrations that differ from those obtained with another platform. 
1201  Therefore, when a new or alternative analytical  platform is replacing a previous platform used 
1202  in the development  of a drug it is important that the potential  differences are well understood. 
1203  The data generated from the previous platform/technology  should be cross validated to that of 
1204  the new or alternative platform/technology.  Seeking feedback from the regulatory authorities is 
1205  encouraged  early in  drug  development.  The use of two  methods  or technologies  within  a 
1206  comparative BA/BE study is strongly discouraged. 
1207  The use of new technology in regulated bioanalysis  should be supported by acceptance criteria 
1208  established a priori based on method development and verified in validation.   
1209  7.6.1 Dried Matrix Methods   
1210  Dried matrix methods (DMM) is a sampling methodology  that offers benefits such as collection 
1211  of reduced blood  sample volumes as a microsampling  technique for drug analysis and ease of 
1212  collection,  storage and transportation.  In addition  to the typical methodological  validation  for 
1213  LC-MS or LBA, use of DMM necessitates further validation  of this sampling  approach before 
1214  using DMM in studies that support a regulatory application,  such as:   
1215    Haematocrit (especially for spotting of whole blood into cards) 
1216    Sample homogeneity  (especially for sub-punch of the sample on the card/device) 
1217    Reconstitution  of the sample 
41 
 
  ICH M10 Guideline 
 
 
1218    DMM sample collection  for ISR 
1219  o  Care should  be taken to ensure sufficient  sample volumes  or numbers of 
1220  replicates are retained for ISR  
1221  o  Should be assessed by multiple  punches of the sample or samples should be 
1222  taken in duplicate   
1223  When DMM is used for clinical  or nonclinical  studies in addition  to typical liquid  approaches 
1224  (e.g., liquid  plasma samples) in the same studies, these two methods should be cross validated 
1225  as described (Refer to Section 6.2). For nonclinical  TK studies, refer to Section 4.1 of ICH S3A 
1226  Q&A. Feedback  from  the  appropriate  regulatory  authorities  is  encouraged  in  early  drug 
1227  development. 
1228  8. DOCUMENTATION     
1229  General  and specific  SOPs and good  record keeping  are essential  to a properly  validated 
1230  analytical  method.  The  data  generated  for  bioanalytical  method  validation  should  be 
1231  documented and available  for data audit and inspection.  Table 1 describes the recommended 
1232  documentation  for submission  to the regulatory authorities  and documentation  that should  be 
1233  available at the analytical site at times of inspection.  This documentation  may be stored at the 
1234  analytical  site or at another secure location.  In this case the documentation  should be readily 
1235  available when requested. 
1236  All  relevant documentation  necessary for reconstructing  the study as it  was conducted and 
1237  reported should be maintained  in a secure environment. Relevant documentation  includes,  but 
1238  is not limited  to, source data, protocols and reports, records supporting procedural, operational, 
1239  and environmental  concerns and correspondence records between all involved  parties.     
1240  Regardless  of  the  documentation  format  (i.e.,  paper  or  electronic),  records  should  be 
1241  contemporaneous with the event and subsequent alterations should not obscure the original  data. 
1242  The basis for changing  or reprocessing data should be documented with sufficient detail, and 
1243  the original  record should  be maintained. Transcripts/copies  of data derived from analyses in 
1244  biohazardous  areas should be maintained if applicable. 
1245  8.1 Summary Information     
1246  Summary information  should include the following  items in Section 2.6.4/2.7.1 of the CTD or 
1247  reports: 
42 
 
  ICH M10 Guideline 
 
 
1248    A summary of assay methods used for each study should be included. Each summary 
1249  should provide the protocol number, the assay type, the assay method identification 
1250  code, the Bioanalytical  Report code, effective date of the method, and the associated 
1251  Validation  Report codes. 
1252    A summary table of all the relevant Validation  Reports should be provided  for each 
1253  analyte, including  Partial Validation  and Cross Validation  Reports. The table should 
1254  include  the assay method identification  code, the type of assay, the reason for the 
1255  new method  or additional  validation  (e.g., to lower the limit  of quantification). 
1256  Changes made to the method should be clearly identified. 
1257    A summary table cross-referencing multiple  identification  codes should be provided 
1258  when an assay has different codes for the assay method, the Validation  Reports and 
1259  the Bioanalytical  Reports. 
1260    Discussion of method changes in the protocol (e.g., evolution  of methods, reason(s) 
1261  for revisions,  unique aspects) 
1262    For comparative BA/BE studies a list of regulatory site inspections including  dates 
1263  and outcomes for each analytical site if available.   
1264  8.2 Documentation for Validation and Bioanalytical Reports    
1265  Table 1 describes the recommended documentation  for the Validation  and Bioanalytical  Reports.
43 
 
   
1266   
Table 1: Documentation and Reporting 
Items  Documentation at the Analytical Site  Validation Report*  Bioanalytical Report* 
Chromatographic    Dates, times, and samples used for    Not applicable    Not applicable 
System Suitability  suitability  testing 
Synopsis    History/evolution  of methods  (e.g.,    Not applicable        Not applicable 
to explain  revisions,  unique  aspects 
Overview of Method 
with supportive data, if available) 
Evolution 
Reference Standards    CoA  or  equivalent  alternative  to    A  copy  of  the  CoA  or    A copy  of  the CoA  or equivalent 
ensure  quality  (including  purity),  equivalent  alternative  alternative  including  batch  /lot 
 
stability/expiration/retest  date(s),  including  batch/lot  number,  number,  source,  quality  (including 
batch number,  and manufacturer or  source,  quality  (including  purity),  storage  conditions,  and 
source  purity),  storage  conditions,  expiration/retest date or a table with 
and expiration/retest  date, or  this information.   
  Log  records  of  receipt,  use,  and 
table with this information. 
storage conditions.    If expired,  quality  and  stability  at 
  If  expired,  quality  and  the time of use and retest dates and 
  If expired, recertified CoA, or retest 
stability  at the time of use and  retested values.   
of quality  and  identity  with  retest 
retest  dates  and  retested 
dates 
values. 
Internal Standard    Name of reagent or standard    Name of reagent or standard 
  IS  quality  or  demonstration  of 
  Origin    Origin 
suitability   
  Log  records  of  receipt,  use,  and 
storage conditions 
1267     
44 
 
 
 
   
Table 1 continued:  Documentation and Reporting 
Items  Documentation at the Analytical Site  Validation Report*  Bioanalytical Report* 
Critical Reagents    Name of reagent    Name of reagent    Name of reagent 
  Batch/ Lot number    Batch/ Lot number    Batch/ Lot number 
  Source/Origin    Source/ Origin    Source/ Origin 
  Concentration, if applicable    Retest date (expiry date)    Retest date (expiry date) 
  Retest date (expiry date)    Storage conditions         Storage conditions      
  Storage conditions            
Stock Solutions    Log  of  preparation,  and  use  of  stock    Notation  that  solutions  were    Notation  that  solutions  were 
solutions  used within stability  period  used within stability  period 
  Storage location and condition    Stock solution  stability      Stock solution  stability   
    Storage conditions    Storage conditions   
Blank Matrix    Records  of  matrix  descriptions,  lot    Description, lot number, receipt    Description,  lot  number, 
numbers, receipt dates, storage conditions,  dates   receipt dates 
and source/supplier 
Calibration    Records and date of preparation    Description  of  preparation    Description of preparation 
Standards  and  including  matrix 
  Record of storage temperature (e.g., log of    Preparation dates and stability 
QCs 
in/out  dates,  analyst,  temperatures,  and    Batch  number,  preparation  period   
freezer(s))  dates and stability  period 
  Storage conditions     
  Storage  conditions 
(temperatures,  dates,  duration, 
etc.)  
 
1268     
45 
 
 
 
   
Table 1 continued:  Documentation and Reporting 
Items  Documentation at the Analytical Site  Validation Report*  Bioanalytical Report* 
SOPs  SOPs for all aspects of analysis, such as:    A  detailed  description  of  the    A  list  of  SOPs/analytical 
assay procedure  protocols  used  for  the  assay 
  Method/procedure 
procedure 
(validation/analytical)   
 
  Acceptance  criteria  (e.g.,  run, 
calibration  curve, QCs)  
  Instrumentation 
  Reanalysis 
  ISR 
  Record  of  changes  to  SOP  (change, 
date, reason, etc.) 
Sample Tracking    Study  sample  receipt,  and  condition  on    Not applicable    Dates of receipt  of shipments 
receipt  number  of  samples,  and  for 
comparative  BA/BE  studies 
  Records that indicate  how samples  were 
the subject ID 
transported  and  received.  Sample 
inventory and reasons for missing samples    Sample condition  on receipt   
  Location of storage (e.g., freezer unit)    Analytical  site  storage 
condition  and location 
  Tracking  logs  of  QCs,  calibration 
standards, and study samples    Storage:  total  duration  from 
sample collection  to analysis 
  Freezer logs for QCs, calibration standards, 
and study samples entry and exit    List  of  any  deviations  from 
planned  storage  conditions, 
and potential  impact 
1269     
46 
 
 
 
   
Table 1 continued:  Documentation and Reporting 
Items  Documentation at the Analytical Site  Validation Report*  Bioanalytical Report* 
Analysis    Documentation  and  data  for  system    Table of all runs (including  failed    Table of all runs, status (accepted 
suitability  checks for chromatography  runs), and analysis dates  and failed), reason for failure, and 
analysis dates. 
  Instrument  use  log,  including  dates  of    Instrument  ID  for  each  run  in 
analysis for each run  comparative BA/BE studies     Instrument  ID  for  each  run  in 
comparative BA/BE studies 
  Sample  extraction  logs  including    Table  of  calibration  standard 
documentation  of  processing  of  concentration  and  response    Table  of  calibration  standard 
calibration  standards,  QCs,  and  study  functions  results  (calibration  concentration  and  response 
samples for each run, including  dates of  curve parameters) of all accepted  function  results (calibration  curve 
extraction  runs with accuracy and precision.  parameters) of  all  accepted runs 
with accuracy and precision.   
  Identity of QCs and calibration  standard    Table of within- and between- run 
lots, and study samples in each run  QC  results  (from  accuracy and    Table  of  QCs  results  of  all 
precision  runs).  Values  outside  accepted runs with  accuracy and 
  Documentation  of  instrument  settings 
should be clearly marked.    precision  results  of the QCs and 
and maintenance 
between-run  accuracy  and 
  Include  total  error  for  LBA 
  Laboratory  information  management  precision  results  from  accepted 
methods 
system (LIMS)  runs.   
  Data  on  selectivity  (matrix 
  Validation  information,  including    Table  of  reinjected  runs  with 
effect), specificity  (interference), 
documentation  and data for:  results  from  reinjected  runs  and 
dilution  linearity  and  sensitivity 
o  Selectivity,  (matrix  effects),  reason(s) for reinjection 
(LLOQ),  carry-over,  recovery.   
specificity,  (interference) 
Bench-top,  freeze-thaw,  long-   QCs  graphs  trend  analysis 
sensitivity,  precision  and 
term, extract, and stock solution  encouraged 
accuracy,  carry-over,  dilution, 
stability 
recovery, matrix effect    Study concentration results table. 
o  Bench-top,  freeze-thaw,  long-   Partial/cross-validation,  if 
  For  comparative  BA/BE studies, 
term, extract, and stock solution  applicable 
IS  response  plots  for  each 
stability 
  Append  separate  report  for  analytical  run,  including  failed 
o  Cross/partial  validations,  if 
additional  validation,  if any    runs   
applicable 
 
47 
 
 
 
   
1270   
Table 1 continued:  Documentation and Reporting 
Items  Documentation at the Analytical Site  Validation Report*  Bioanalytical Report* 
Chromatograms    Electronic audit trail:      Representative    For and comparative BA/BE studies, 
and Reintegration  chromatograms  (original  100% of chromatograms.   
  100% e-chromatograms of original  and 
and reintegration) 
reintegration  from  accepted  and  fail    Chromatograms  may be submitted  as 
runs    Reason for reintegration  a supplement 
  Reason for reintegration    For  comparative  BA/BE    For  comparative  BA/BE  studies, 
studies,  100%  original  and  reintegrated 
  Mode  of  reintegration100%  of  run 
chromatograms  of  original  chromatograms and initial  and repeat 
summary sheets of accepted and failed 
and  reintegration  from  integration  results 
runs,  including  calibration  curve, 
accepted and fail runs.   
regression,  weighting  function,  analyte    For other studies,  randomly  selected 
and  IS  response  and  retention  time,    Chromatograms  may  be  chromatograms  from  5%  of  studies 
response ratio, integration  type  submitted as a supplement  submitted in application  dossiers 
    For  comparative  BA/BE    Reason for reintegration 
studies,100%  of  run 
  Identification  and  discussion  of 
summary sheets of accepted 
chromatograms  with  manual 
and  failed  runs,  including 
reintegration 
calibration  curve, 
regression,  weighting    SOP for reintegration,  as applicable 
function,  analyte  and  IS 
  For  comparative  BA/BE  studies, 
responses  and  retention 
100%  of  run  summary  sheets  of 
times  and  dilution  factor if 
accepted  and  failed  runs,  including 
applicable. 
calibration  curve,  regression, 
  weighting  function,  analyte  and  IS 
responses  and  retention  times,  and 
dilution  factor if applicable. 
1271     
48 
 
 
 
   
Table 1 continued:  Documentation and Reporting 
Items  Documentation at the Analytical Site  Validation Report*  Bioanalytical Report* 
Deviations from Procedures    Contemporaneous  documentation  of    Description  of    Description of deviations 
deviations/  unexpected events  Deviations 
  Impact on study results 
  Investigation  of unexpected events    Impact on study results 
  Description  and  supporting 
  Impact assessment    Description  and  data  of  significant 
supporting  data  of  investigations 
significant investigations 
Repeat Analysis    SOP  for  conducting  reanalysis/repeat    Not applicable    Table of sample IDs, reason for 
analysis (define reasons for reanalysis, etc.)  reassay,  original  and  reassay 
values,  reason  for  reported 
  Retain 100% of repeat/reanalysed data 
values, run IDs  
  Contemporaneous  records  of  reason  for 
  Reanalysis SOP, if requested   
repeats 
ISR    SOP for ISR    Not applicable    ISR  data  table  (original  and 
reanalysis  values and run IDs, 
  ISR  data:  Run  IDs,  run  summary  sheets, 
percent  difference,  percent 
chromatograms  or  other  electronic 
passed) 
instrument data files 
  ISR  failure  investigations,  if 
  Document ISR failure investigations,  if any 
any 
  SOP for ISR (if requested) 
Communication    Between  involved  parties  (Applicant,    Not applicable    Not applicable 
contract research organizations  (CROs), and 
consultants) related to study/assay 
Audits and Inspections    Audit and inspection  report    Not applicable    Not applicable 
1272  *The applicant  is expected to maintain  data at the analytical  site to support  summary data submitted  in Validation  and Bioanalytical  Reports.   
1273  Validation  and Bioanalytical  Reports should be submitted in the application. 
49 
 
 
 
   
1274   May append or link from Validation  Report. 
1275  Submit either in Validation  Report or in Bioanalytical  Report
50 
 
 
 
   
1276  9. GLOSSARY    
1277  Accuracy:   
1278  The degree of closeness of the measured value  to the nominal  or known  true value  under 
1279  prescribed conditions  (or as measured by a particular  method). In this document  accuracy is 
1280  expressed as percent relative error of the nominal  value. 
1281  Accuracy (%) = ((Measured Value-Nominal  Value)/Nominal  Value)  100 
1282   
1283  Analysis:  
1284  A series of  analytical  procedures  from  sample  processing/dilution  to measurement  on  an 
1285  analytical instrument. 
1286   
1287  Analyte:   
1288  A specific chemical  moiety  being  measured,  including  an intact drug,  a biomolecule  or its 
1289  derivative or a metabolite in a biologic  matrix. 
1290   
1291  Analytical Procedure:  
1292  The analytical  procedure refers to the way of performing  the analysis. It should  describe in 
1293  detail the steps necessary to perform each analysis. 
1294   
1295  Analytical Run (also referred to as Run):  
1296  A complete set of analytical and study samples with appropriate number of calibration standards 
1297  and QCs for their validation.  Several runs may be completed in one day or one run may take 
1298  several days to complete.    
51 
 
 
 
   
1299  Anchor Calibration Standards/Anchor Points:  
1300  Spiked samples set at concentrations below the LLOQ or above the ULOQ of the calibration 
1301  curve and analysed to improve curve fitting in LBAs. 
1302   
1303  Batch (for Bioanalysis): 
1304  A batch is comprised of QCs and study samples which are handled during a fixed period of time 
1305  and by the same group of analysts with the same reagents under homogenous conditions. 
1306   
1307  Batch (for Reference Standards and Reagents): 
1308  A specific quantity of material produced in a process or series of processes so that it is   
1309  expected to be homogeneous within  specified limits. Also referred to as Lot.   
1310   
1311  Biological Drugs: 
1312  Drugs manufactured by using  biotechnology  (e.g., therapeutic  proteins). Also referred to as 
1313  large molecule drugs.   
1314   
1315  Biological Matrix:  
1316  A biological  material including,  but not limited  to, blood, serum, plasma and urine. 
1317   
1318  Binding Reagent:  
1319  A reagent that directly binds to the analyte in LBA-based bioanalytical  methods. 
1320   
1321  Blank Sample:   
1322  A sample of a biological  matrix to which no analyte and no IS has been added. 
52 
 
 
 
   
1323  Calibration Curve:    
1324  The relationship  between the instrument  response (e.g., peak area, height  or signal)  and the 
1325  concentration  (amount)  of analyte  in the sample  within  a given  range. Also  referred to as 
1326  Standard Curve.   
1327   
1328  Calibration Range:  
1329  The calibration  range of an analytical  procedure is the interval  between the upper and lower 
1330  concentration (amounts) of analyte in the sample (including  these concentrations) for which it 
1331  has been demonstrated  that the analytical  procedure meets the requirements  for precision, 
1332  accuracy and response function. 
1333   
1334  Calibration Standard:   
1335  A matrix to which a known amount of analyte has been added or spiked. Calibration  standards 
1336  are used to construct calibration  curves. 
1337   
1338  Carry-over:   
1339  The appearance of an analyte signal in a sample from a preceding sample.   
1340   
1341  Chemical Drugs: 
1342  Chemically  synthesised drugs. Also referred to as small molecule drugs. 
1343   
1344  Critical Reagent:  
1345  Critical  reagents  for  LBAs  include  binding  reagents  (e.g.,  antibodies,  binding  proteins, 
1346  peptides) and those containing  enzymatic  moieties that have a direct impact on the results of 
1347  the assay.   
53 
 
 
 
   
1348  Cross Validation:   
1349  Comparison  of  two  bioanalytical  methods  or  the  same  bioanalytical  method  in  different 
1350  laboratories in order to demonstrate that the reported data are comparable. 
1351   
1352  Dilution Integrity:  
1353  Assessment of the sample dilution  procedure to confirm that the procedure does not impact the 
1354  measured concentration of the analyte. 
1355   
1356  Dilution Linearity:   
1357  A  parameter  demonstrating  that  the  method  can  appropriately  analyse  samples  at  a 
1358  concentration exceeding the ULOQ of the calibration  curve without influence of hook effect or 
1359  prozone  effect and that the measured concentrations  are not affected by dilution  within  the 
1360  calibration  range in LBAs. 
1361   
1362  Full Validation:     
1363  Establishment of all validation  parameters that ensure the integrity of the method when applied 
1364  to sample analysis. 
1365   
1366  Hook Effect:  
1367  Suppression of response due to very high concentrations of a particular analyte. A hook effect 
1368  may occur in LBAs that use a liquid-phase  reaction step for incubating  the binding  reagents 
1369  with the analyte. Also referred to as prozone. 
1370   
1371  Incurred Sample:   
1372  A sample obtained from study subjects or animals.    
54 
 
 
 
   
1373  Incurred Sample Reanalysis (ISR):    
1374  Reanalysis of a portion  of the incurred samples in a separate analytical  run on a different day 
1375  to determine whether the original  analytical results are reproducible.   
1376   
1377  Interfering Substance:  
1378  A substance that is present in the matrix that may affect the analysis of an analyte. 
1379   
1380  Internal Standard (IS):   
1381  A structurally  similar  analogue  or  stable  isotope  labelled  compound  added  to  calibration 
1382  standards,  QCs  and  study  samples  at  a  known  and  constant  concentration  to  facilitate 
1383  quantification  of the target analyte. 
1384   
1385  Ligand Binding Assay (LBA):  
1386  A method to analyse an analyte of interest using reagents that specifically  bind to the analyte. 
1387  The analyte is detected using reagents labelled  with e.g. an enzyme, radioisotope,  fluorophore 
1388  or chromophore. Reactions are carried out in microtitre plates, test tubes, disks, etc. 
1389   
1390  Lower Limit of Quantification (LLOQ):  
1391  The lowest  amount  of an analyte  in  a sample  that can be quantitatively  determined  with 
1392  predefined precision and accuracy. 
1393   
1394  Matrix Effect:   
1395  The direct or indirect  alteration or interference in response due to the presence of unintended 
1396  analytes or other interfering substances in the sample.   
55 
 
 
 
   
1397  Method:  
1398  A comprehensive description  of all procedures used in sample analysis. 
1399   
1400  Minimum Required Dilution (MRD):  
1401  The initial  dilution  factor by which biological  samples are diluted  with buffer solution  for the 
1402  analysis  by  LBAs. The MRD may  not  necessarily  be the ultimate  dilution  but  should  be 
1403  identical  for all  samples  including  calibration  standards and  QCs. However,  samples  may 
1404  require further dilution. 
1405   
1406  Nominal Concentration:  
1407  Theoretical or expected concentration. 
1408   
1409  Parallelism:     
1410  Parallelism  demonstrates that the serially diluted  incurred sample response curve is parallel to 
1411  the calibration  curve. Parallelism is a performance characteristic that can detect potential matrix 
1412  effects.  
1413   
1414  Partial Validation:   
1415  Evaluation  of modifications  to already fully validated analytical methods.   
1416   
1417  Precision:    
1418  The closeness of agreement (i.e., degree of scatter) among a series of measurements. Precision 
1419  is  expressed as the coefficient  of variation  (CV) or the relative  standard deviation  (RSD) 
1420  expressed as a percentage. 
1421  Precision (%) = (Standard Deviation / Mean) x 100 
56 
 
 
 
   
1422  Processed Sample:  
1423  The final  sample that has been subjected to various manipulations  (e.g., extraction, dilution, 
1424  concentration). 
1425     
1426  Quality Control Sample (QC):  
1427  A sample spiked with a known quantity of analyte that is used to monitor the performance of a 
1428  bioanalytical  method and assess the integrity and validity  of the results of the unknown samples 
1429  analysed in an individual  batch or run.   
1430   
1431  Recovery:  
1432  The extraction efficiency of an analytical process, reported as a percentage of the known amount 
1433  of an analyte carried through the sample extraction and processing steps of the method.   
1434   
1435  Reproducibility:   
1436  The extent to which consistent results are obtained when an experiment is repeated.  
1437   
1438  Response Function:    
1439  A function which adequately describes the relationship  between instrument response (e.g., peak 
1440  area or height ratio or signal) and the concentration (amount) of analyte in the sample. Response 
1441  function is defined within a given range. See also Calibration  Curve. 
1442   
1443  Selectivity:  
1444  Ability  of an analytical  method  to differentiate  and measure the analyte in  the presence of 
1445  interfering substances in the biological  matrix (non-specific interference).    
57 
 
 
 
   
1446  Sensitivity:  
1447  The lowest analyte concentration that can be measured with acceptable accuracy and precision 
1448  (i.e., LLOQ). 
1449   
1450  Specificity:   
1451  Ability  of an analytical  method to detect and differentiate the analyte from other substances, 
1452  including  its related substances (e.g., substances that are structurally  similar  to the analyte, 
1453  metabolites,  isomers, impurities  or concomitant medications). 
1454   
1455  Standard Curve:  
1456  The relationship  between the instrument  response (e.g., peak area, height  or signal)  and the 
1457  concentration  (amount)  of analyte  in the sample  within  a given  range. Also  referred to as 
1458  calibration  Curve.   
1459   
1460  Standard Operating Procedure (SOP):  
1461  Detailed written instructions  to achieve uniformity  of the performance of a specific function. 
1462   
1463  Surrogate Matrix:  
1464  An alternative to a study matrix of limited  availability  (e.g., tissue, cerebrospinal fluid,  bile) or 
1465  where the study matrix contains an interfering endogenous counterpart. 
1466   
1467  System Suitability:   
1468  Determination  of instrument performance (e.g., sensitivity  and chromatographic  retention) by 
1469  analysis of a set of reference standards conducted prior to the analytical run.    
58 
 
 
 
   
1470  Total Error:     
1471  The sum of the absolute value of the errors in accuracy (%) and precision  (%). Total error is 
1472  reported as percent (%) error. 
1473   
1474  Upper Limit of Quantification (ULOQ):  
1475  The upper limit  of quantification  of an individual  analytical procedure is the highest amount of 
1476  analyte  in  a sample  that can be quantitatively  determined  with  pre-defined  precision  and 
1477  accuracy. 
1478   
1479  Validation:   
1480  Demonstration that a bioanalytical  method is suitable for its intended purpose. 
1481   
1482  Working Solution:   
1483  A non-matrix  solution  prepared by diluting  the stock solution  in an appropriate solvent. It is 
1484  mainly added to matrix to prepare calibration  standards and QCs. 
1485   
1486  Zero Sample:   
1487  A blank sample spiked with an IS. 
59