This document discusses strategies for whole blood sample analysis using LC-MS/MS for bioanalysis. It reviewed over 1000 bioanalytical methods and found that 90% met acceptance criteria for whole blood stability testing, while 10% failed. Three case studies are presented demonstrating different analyte stability in whole blood and plasma under various temperature and processing conditions. Proper sample handling and treatment are important to obtain an accurate analysis and meet acceptance criteria for whole blood stability testing.

1. Presented at ASMS 2019
Whole Blood Sample Analysis Strategies for LC-MS/MS Approach Bioanalysis
Yongle Pang1, Theodore Brus2, Anita Wyeth1 and Stephanie Cape1
1Covance Laboratories Inc., Madison, WI; 2Covance Laboratories Inc., Indianapolis, IN
Introduction
LC-MS/MS is widely used in bioanalysis, which is focused on
the quantitative analysis of molecules of interest in biological
matrices. A reliable bioanalytical method should reveal the
drug concentration at the moment of sample collection. Based
on FDA and EMA guidelines, the stability of the analyte should
be assessed from sample collection to analysis, and proper
treatment, if needed, should be applied to the samples for an
accurate analysis. Based on a review of 1000+ studies
validated, 90% met all acceptance criteria for whole blood
stability (WBS) testing. 10% of methods failed to meet the
criteria. 47% of these also showed plasma instability. Although
in vitro WBS experiments cannot absolutely mimic sample
collection, it is important that the stability of the analyte in
whole blood is appropriately considered.
Figure 1. General procedures for whole blood stability test.
Instrumentation
LC: Shimadzu, Prominence, 20/30 Series
MS: Sciex API 4000/5000/5500/6500 LC-MS/MS system
Method
▶ Prepare QC in WB and mix gently for at least 2 minutes.
▶ Incubate the spiked sample in a water bath at 37°C for at
least 10 minutes to equilibrate.
▶ Gently mix the spiked blood for a further 30 seconds and
divide into aliquots as required.
▶ Harvest plasma from T0 samples immediately, and store half
the remaining aliquot(s) at room temperature (RT) and
another half on wet ice (WI).
▶ After 1h or 2h incubation, harvest plasma from aliquots from
each temperature at each timepoint.
▶ Peak area ratio of the T1 and T2 samples will be compared
with T0 sample.
Results and Discussion
▶ Data from 1076 validated plasma methods was assessed.
▶ 90% of the assays passed WBS test, while 10% of cases,
WBS failed either 2h RT or 2h on WI.
▶ In 55 studies, the analyte(s) that failed the WBS test was
unstable in plasma, and in 49 cases, the analyte(s) that
failed the WBS test was stable in plasma.
▶ Analyte is defined as “unstable in plasma” if it needs sample
treatment, WI handling or has less than 24 h RT plasma
stability.
▶ WBS is considered acceptable if the concentration or the
mean peak area ratio of the stored sample is within ±15.0%
of the T0 sample.
Figure 2. Bar graph shows the percentage of the passed and failed whole
blood stability tests. Pie graph shows the percentage of pass/fail results at RT
or on WI in 49 methods which WBS failed but RT plasma stability is good.
Case Study I: Determination of Bendamustine and gamma-
Hydroxybendamustine in Human Plasma by LC-MS/MS
▶ Both analytes had stability issues in plasma at RT, but matrix
stability data on WI met acceptance criteria.
▶ WBS data shows a decreasing trend with longer incubation
time at RT.
▶ Whole blood samples should be incubated on WI
immediately after sample collection.
Figure 3. Structure of Bendamustine (A) and gamma-Hydroxybendamustine (B).
N
N
N
ClCl
OH
O
H Cl
N
N
N
ClCl
ONa
O
HO
(A) (B)
Table 1. WBS Data for Bendamustine (top) and gamma-
Hydroxybendamustine (bottom) in Human Whole Blood after
Storage at Room Temperature or Wet Ice Conditions
Case Study II: Determination of Bortezomib in Human
Plasma by LC-MS/MS
▶ The assay failed both RT and WI stability in plasma.
▶ Matrix stability passed when handling the samples on WI
with formic acid treatment.
▶ Whole blood QC was prepared with acidified whole blood.
▶ The accuracy was 52% after incubation at RT for 0.5h, while
the accuracy met acceptance criteria (104.8%) on WI with
incubation for 0.5h.
▶ Whole blood samples should be treated with formic acid
immediately after sample collection and incubated on WI for
no longer than 30 mins.
N
N
N
H
O
H
N B
OH
OH
O
Figure 4. Structure of Bortezomib.
Table 2. WBS Data for Bortezomib in Human Whole Blood
after Storage at Room Temperature or Wet Ice Conditions
Conclusion
▶ WBS test failure is often due to partitioning.
▶ Factors impacting the partitioning include time, hematocrit,
temperature, hemolysis, drug concentration, age of blood,
disease state and anticoagulant.
▶ In the absence of plasma instability, whole blood processing
at room temperature within 2 hours was demonstrated to be
appropriate >97% of the time.
Case Study III: Determination of Atorvastatin, p-Hydroxy
Atorvastatin and o-Hydroxy Atorvastatin in Human
Plasma by LC-MS/MS
▶ All three analytes showed good RT stability in plasma.
▶ Atorvastatin and p-hydroxy atorvastatin were stable in
human whole blood for 2h RT and 2h WI.
▶ o-Hydroxy atorvastatin passed 2h RT WBS, but failed 2h
WI WBS.
▶ The WBS failure for o-hydroxy atorvastatin was highly
possible due to temperature dependent partitioning.
▶ Whole blood samples should be collected at RT.
Figure 5. Structure of Atorvastatin (A), p-Hydroxy Atorvastatin (B) and
o-Hydroxy Atorvastatin (C).
N
H
O
N
F
OH
CO2
OH
Ca2+
2
N
H
O
N
F
OH
CO2Na
OH
NaO
OH
N
H
O
N
F
OH
CO2Na
OH
2H2O
(A) (B)
(C)
(A) (B)
(C)
Table 3. WBS Data for Atorvastatin (A), p-Hydroxy Atorvastatin
(B) and o-Hydroxy Atorvastatin (C)in Human Whole Blood after
Storage at Room Temperature or Wet Ice Conditions