Evaluation of Internal 
 
Standard Responses During 
Chromatographic 
Bioanalysis: Questions and 
Answers 
Guidance for Industry 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
 
 
September 2019 
Biopharmaceutics
   
Evaluation of Internal 
Standard Responses During 
Chromatographic 
Bioanalysis: Questions and 
 
Answers
Guidance for Industry 
 
 
 
 
Additional copies are available from: 
 
Office of Communications, Division of Drug Information  
Center for Drug Evaluation and Research 
Food and Drug Administration 
10001 New Hampshire Ave., Hillandale Bldg., 4th Floor  
Silver Spring, MD 20993-0002  
Phone: 855-543-3784 or 301-796-3400; Fax: 301-431-6353 
Email: druginfo@fda.hhs.gov  
https://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
 
 
 
September 2019 
Biopharmaceutics 
      Contains Nonbinding Recommendations 
 
TABLE OF CONTENTS 
 
 
 
I.  INTRODUCTION............................................................................................................. 1 
II.  QUESTIONS AND ANSWERS ....................................................................................... 2 
i  Contains Nonbinding Recommendations 
 
Evaluation of Internal Standard Responses during 
Chromatographic Bioanalysis: Questions and Answers 
Guidance for Industry1 
 
 
This guidance represents 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 office responsible for this guidance as listed on the title 
page.  
 
 
 
I.  INTRODUCTION  
 
This guidance provides recommendations to sponsors, applicants, and contract research 
organizations regarding internal standard (IS) response 2 variability in chromatographic 
analytical data submitted in investigational new drug applications, new drug applications, 
abbreviated new drug applications, biologics license applications, and supplements. 
Chromatographic analytical methods are commonly used to quantitate analyte concentrations in 
samples from nonclinical and clinical studies to support regulatory submissions. Depending upon 
its source, IS response variability may impact the accuracy of analyte concentration 
measurements. This question and answer (Q&A) document provides the Agencys current 
thinking on IS response variability and its potential impact on the accuracy of analyte 
concentration measurements. This Q&A also suggests approaches to determine whether 
observed IS response variability is likely to impact the accuracy of the data such that further 
investigation into the root cause(s) is warranted. 
 
In general, FDAs guidance documents do not establish legally enforceable responsibilities. 
Instead, guidances describe the Agencys current thinking on a topic and should be viewed only 
as recommendations, unless specific regulatory or statutory requirements are cited. The use of 
the word should in Agency guidances means that something is suggested or recommended, but 
not required.  
 
 
 
 
                                                 
1 This guidance has been prepared by the Office of Study Integrity and Surveillance, the Office of Generic Drugs, 
and the Office of Clinical Pharmacology in the Center for Drug Evaluation and Research at the Food and Drug 
Administration. You may submit comments on this guidance at any time. Submit comments to Docket No. FDA-
2017-D-6821 (available at https://www.regulations.gov/docket?D=FDA-2017-D-6821). See the instructions in that 
docket for submitting comments on this and other Level 2 guidances. 
 
2 An IS response is a measurement of the signal from the IS, which is typically generated during liquid 
chromatography-mass spectrometry bioanalysis. 
  1  Contains Nonbinding Recommendations 
 
II.  QUESTIONS AND ANSWERS 
 
Q1: What are internal standards in analytical chemistry, and why are they used in 
bioanalysis? 
 
A1: In analytical chemistry, ISs are usually a structural analog or stable isotope of an analyte of 
interest and are commonly used in chromatographic analytical methods to correct for variability 
in sample processing and analysis. In chromatographic bioanalysis, an IS is added to all samples, 
including calibration standards (Cals), quality controls (QCs), and subject samples prior to 
extraction. The specific IS selected for a method should have similar physicochemical properties 
to the analyte of interest so that it behaves in a similar fashion to the analyte and reflects any 
changes to the analyte measurement that may occur during sample processing and analysis.  
 
Q2: What are sources of IS response variability?  
 
A2: By design, the intention is to add the same amount of IS to all samples. However, variability 
in IS responses among samples is occasionally observed, even among samples analyzed in the 
same run. Potential sources of IS response variability include human errors made during sample 
preparation or processing, instrumental issues that may occur during analysis, and matrix effects. 
   
Q3: When is IS response variability not likely to impact the accuracy of the data? 
 
A3: IS response variability is not likely to impact the accuracy of the data when the range of IS 
responses for subject samples is similar to the range of IS responses for Cals/QCs in the same 
analytical run. For example, variability in the IS responses for subject samples is not likely to 
impact the accuracy of the data under the following circumstances: 
 
Example 1: IS responses for subject samples are similar to IS responses observed for Cals/QCs 
in the same analytical run (see Figure 1). 
   
  2  Contains Nonbinding Recommendations 
 
Figure 1. Plot of IS responses in an analytical run, exemplifying IS responses for subject 
samples that are similar to IS responses observed for Cals/QCs 
 
5.E+07
4.E+07
s Cals
nt
u
o 3.E+07 QCs
C
ea  Blanks
Ar
k  Subject 1
a
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S P 2.E+07 Subject 2
I
1.E+07
0 4 8 12 16 20 24 28 32 36 40 44 48 52
Injection Number
 
 
Example 2: IS responses for subject samples demonstrate a gradual drift or repeating pattern, 
but the range of IS responses for subject samples is similar to the range of IS responses for 
Cals/QCs in the same run (see Figure 2).  
 
Figure 2. Plot of IS responses in an analytical run, exemplifying IS responses for subject 
samples demonstrating a gradual drift or repeating pattern similar to Cals/QCs 
 
5.E+07
4.E+07
s Cals
nt
u
o 3.E+07 QCs
C
ea  Blanks
Ar
k  Subject 1
a
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S p 2.E+07 Subject 2
I
1.E+07
0 4 8 12 16 20 24 28 32 36 40 44 48 52
Injection Number
 
 
 
  3  Contains Nonbinding Recommendations 
 
Q4: When can IS response variability impact the accuracy of the data? 
 
A4: IS response variability may impact the accuracy of the data when the range of the IS 
responses for subject samples is different than the range of IS responses for Cals/QCs in the 
same run. For example, IS response variability for subject samples may impact the accuracy of 
the data under the following circumstances:  
 
Example 1: IS responses for one or more subject samples are substantially different from the IS 
responses for the majority of the other subject samples, and the IS responses for Cals/QCs do not 
demonstrate a similar variability pattern (see Figure 3). 
 
Figure 3. Plot of IS responses in an analytical run, exemplifying an IS response for a 
subject sample that is substantially different from the responses for the majority of the 
other subject samples and Cals/QCs 
 
9.E+07
8.E+07
7.E+07
6.E+07
s
nt Cals
u
o
C 5.E+07 QCs
a 
e
Ar Blanks
k  4.E+07
a Subject 1
e
P
IS  3.E+07 Subject 2
2.E+07
1.E+07
0 4 8 12 16 20 24 28 32 36 40 44 48 52
Injection Number
 
 
Example 2: There is a gradual increase or decrease extended beyond the range of the IS 
responses for subject samples, and there are no IS responses for QCs interspersed among the 
impacted segment of the run (see Figure 4). 
  4  Contains Nonbinding Recommendations 
 
Figure 4. Plot of IS responses in an analytical run, exemplifying a gradual decrease in IS 
responses for subject 2 samples without IS responses for QCs interspersed among the 
impacted segment of the run 
 
5.E+07
4.E+07
a Cals
e
k Ar 3.E+07 QCs
a
Pe Blanks
S 
I
Subject 1
2.E+07 Subject 2
1.E+07
0 4 8 12 16 20 24 28 32 36 40 44 48 52
Injection Number
 
 
Example 3: IS responses for subject samples are consistently lower or higher than IS responses 
for Cals/QCs (see Figure 5). 
 
Figure 5. Plot of IS responses in an analytical run, exemplifying IS responses for subject 2 
samples that are consistently higher than IS responses for Cals/QCs 
 
8.E+07
7.E+07
6.E+07
s
unt 5.E+07 Cals
o
C QCs
a 
e
Ar 4.E+07 Blanks
k 
ea Subject 1
P
IS  3.E+07 Subject 2
2.E+07
1.E+07
0 4 8 12 16 20 24 28 32 36 40 44 48 52
Injection Number
 
 
  5  Contains Nonbinding Recommendations 
 
Reviewing the scatter plot of IS responses for all samples in the run might help identify 
variability trends that may or may not impact the accuracy of the data.  
 
Q5: How can sponsors address concerns about IS response variability? 
 
A5: When IS responses for subject samples are outside of the ranges observed for Cals/QCs that 
meet the run acceptance criteria, the subject sample concentration measurements might not be 
accurate. Therefore, sponsors should determine whether this observed difference impacts the 
accuracy of the data for subject samples.  
 
For subject samples with IS responses significantly different from the responses for Cals/QCs (as 
pre-defined in a standard operating procedure (SOP)) in a run (see Figure 3), pre-established 
criteria for repeat analysis should be followed. The repeat analysis criteria should be justified and 
the protocol for the number of replicates reanalyzed for each sample and final values reported 
should be established in an SOP before initiation of the study.  
 
For runs where a gradual drift or pattern in IS responses is observed between subject samples and 
there are no IS responses for Cals/QCs within the impacted segment of the run, subject samples 
displaying the aberrant IS responses (see Figure 4) should be reanalyzed. If the IS responses for 
subject samples in the repeat analysis are similar to those of Cals/QCs, and if the analyte 
concentrations in a repeat run are within 20% of the original assay results of the individual 
samples, generally no further investigation into the cause of the initial variability is necessary. 
Sample values to be reported should follow a pre-established procedure. However, if the IS 
responses for subject samples in the repeat analysis are similar to those of Cals/QCs, but the 
analyte concentrations in the repeat analysis are not within 20% of the original assay results of 
the individual samples, then sponsors should report analyte concentrations from the repeat 
analysis. 
 
When IS responses for subject samples are consistently lower or higher than IS responses for 
Cals/QCs (see Figure 5), a subset of subject samples should be reanalyzed. If IS responses of 
subject samples in the repeat analysis are again not within the range for those of Cals/QCs (i.e., 
differences in IS responses in the original run are reproducible), and repeat analyte 
concentrations are still comparable to the original values, additional data might be needed to 
demonstrate that the differences in sample matrices (i.e., matrices for subject samples vs. 
matrices for Cals/QCs) do not impact the accuracy of the analyte concentration measurements. 
The approach(es) for investigation might include, but are not limited to, an analysis of QCs 
prepared in pre-dose matrix of individual impacted subjects, serial dilution of impacted subject 
samples using the matrices used to prepare Cals/QCs, or any other scientifically justified 
approach. 
  6