Analysis of Blood Sample by HPLC
Lab Partners: Adam Woodard, Katrin Henry
TA: Jie Ding
Date: March 27, 2013
Abstract
The HPLC was used to analyze a blood sample for a scenario of a person’s possible cause of
death. The calibration curve and correlation for Disopyramide was y=7882x+52.876,
R2=0.9972; Lidocaine was y=894.x+35.025, R2=0.9999; Procainamide was y=23853x+12.175,
R2=0.8616. The R2 values were very high and showed a very high correlation. The sample
contained Disopyramide, Lidocaine, and Procamide based on retention times. There were toxic
levels of Disopyramide >0.005 (𝜇𝑔/𝜇𝐿) and Lidocaine >0.005 (𝜇𝑔/𝜇𝐿), indicating the person
died of poisoning.
Introduction
High pressure liquid chromatography (HPLC) is useful to identify, quantify, and purify
chemicals. The advantages of the HPLC method are the separation technique, non-destructive to
the samples, and low cost. The competing instrument is the gas liquid chromatography (GLC).
While the GLC can match performance (high speed HPLC of two-minutes), the samples undergo
derivatisation in order to be volatile. The detector of the HPLC is a UV detector and the do not
require samples to be volatile.1 The HPLC has application is in pharmaceutical, toxicology,
medical, and manufacturing industries.
The HPLC uses retention time to identify a chemical in a mixture. A calibration curve was made
for each standard Disopyramide, Procainamide, and Lidocaine. The toxic levels of the chemicals:
Drug Chart
Drug
Disopyramide
Lidocaine
Procainamide
Therapuetic Level
(𝜇𝑔/𝜇𝐿)
0.002-0.005
0.0015-0.005
0.002-0.005
Toxic Level
(𝜇𝑔/𝜇𝐿)
>0.005
>0.005
>0.010
Experimental
(𝜇𝑔/𝜇𝐿)
0.0585
0.0901
0.0065
Linear Range
(𝜇𝑔/𝜇𝐿)
0.01-0.5
0.025-2.5
0.00125-0.04
Experimental Methods
An Agilent HPLC had the experimental parameters: flow rate: 1.0 mL/min; wavelength: 254 nm;
injections: 50 𝜇𝐿. The solvent was a solution of methanol and 0.1% Trifluoro acetic acid (LC
grade). A Hamilton syringe was used for 50 𝜇𝐿 manual injections. All the standards had a
concentration of 2.5 𝜇𝑔/𝐿.
Table 1: Equipment
Equipment
HPLC
Syringe
Model
Agilent 1100
G1314A#PE51530425
50 𝜇𝐿
Results
HPLC Spectra
Comparing Retention Times
Au (Logarithmic)
1000
100
Disopyramide
Procainamide
10
Lidocaine
Unknown
1
0
1
2
3
4
5
6
7
Time (min)
Calibration Curves
Disopyramide, Lidocaine
5000
Disopyramide
y = 7882x + 52.876
R² = 0.9972
3000
Lidocaine
2000
Linear (Disopyramide)
y = 894.3x + 35.025
R² = 0.9999
1000
Linear (Lidocaine)
0
0
0.5
1
1.5
Concentration (g/L)
2
2.5
Procainamide
Peak Area
Peak Area
4000
800
700
600
500
400
300
200
100
0
y = 23853x + 12.175
R² = 0.8616
0
0.005
0.01
0.015
0.02
Concentration (g/L)
0.025
0.03
Discussion
Each peak had a retention time. The first few peaks on the individual spectra were caused by
bubbles from the solvent line and ignored. The exact HPLC procedure was used for the standards
and unknown; therefore, the retention time was used to distinguish between the chemicals.
The analytes were plotted on a single graph. The vertical axis was adjusted to a logarithmic scale
to make the plots easier to read. Additionally, the unknown curve was adjusted lower relative to
the other curves. None of the data was compromised nor did the adjustment change the
interpretation of the retention times. Only the data from the first dilution was plotted, because the
concentration affects the integration under the curve, not the retention time.
The calibration curve and correlation for Disopyramide was y=7882x+52.876, R2=0.9972;
Lidocaine was y=894.x+35.025, R2=0.9999; Procainamide was y=23853x+12.175, R2=0.8616.
The R2 values were very high and showed a very high correlation.
The retention time of Disopyramide was t=5.0717; Lidocaine was t=2.06752; Procainamide was
t=3.7468. The unknown or blood sample was created using the possible standards. Based on
visual comparison of the retention time, the unknown contained all the standards.
The unknown had area under calculated by the NORM.DIST function on Excel. The integration
is not as accurate as the direct measurement from the HPLC, but that data not recorded. The area
under the curve for Disopyramide was 514.168, concentration 0.0585 (𝜇𝑔/𝜇𝐿); Lidocaine was
115.622, concentration 0.0901 (𝜇𝑔/𝜇𝐿); Procainamide was 168.209, concentration 0.0065
(𝜇𝑔/𝜇𝐿).
The analysis of the “blood” sample based on retention times determined that Disopyramide,
Lidocaine, and Procainamide were present. There were toxic levels of Disopyramide >0.005
(𝜇𝑔/𝜇𝐿) and Lidocaine >0.005 (𝜇𝑔/𝜇𝐿), indicating the person died of poisoning. There were
not toxic levels of Procamide.
References
[1] Fallon, R.F.; Booth, L.D.; Applications of HPLC in Biochemistry. Chapter 1, pg 5.
[2] Bridges et al., An HPLC Method for the Simultaneous Quantitation of Quinidine,
Procainamide, N-Acetyprocainamide and Disopyramide, Journal or Analytical Toxicology
(1983) 7, 65-68.
[3] Qualitative HPLC Analysis. George Mason University.
http://www.gmu.edu/depts/SRIF/tutorial/hplc/qual.htm. Accessed March 27, 2013.
[4] Preparation of Calibration Curves.
http://www.nmschembio.org.uk/dm_documents/lgcvam2003032_xsjgl.pdf. Accessed March 27,
2013.