Simple, Fast and Accurate
Quantitation of Human Plasma
Vitamins and Their Metabolites by Protein
Precipitation Combined with Columns Using HPLC-UV, HPLC-FLD or LC/MS/MS
PO13410513_W
Shuguang Li, Jason Anspach, Sky Countryman, and Erica Pike
Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA
Abstract
Monitoring the body’s concentration of vitamins and their
metabolites (e.g. phosphates) is an aid in diagnosing and
treating disease states related to their deficiencies. The
number of laboratories that analyze vitamins and their
metabolites has risen dramatically, a majority of which
are under pressure to rapidly and accurately process patient samples. It is important that the analytical method
be rapid, sensitive, and accurate in order to accommodate
these high-throughput labs that analyze thousands of patient samples each week. Existing analytical methods are
challenging due to the complex nature of human plasma /
serum matrices. In addition, to achieve the required detection limits, samples require complex liquid-liquid extraction techniques followed by derivatization.
Traditionally, a protein precipitation step is used for fast
cleanup of plasma samples. Protein precipitation is normally performed using a centrifuge tube or a 96-well collection plate. However, this process requires that supernatant be collected while being careful not to disrupt the
pelleted protein in the bottom of the tube or collection
plate. This step was greatly simplified by using Impact™
Protein Precipitation Plates.
Three examples will be given in this poster. Vitamins and /
or their metabolites were extracted from human plasma by
performing a rapid protein precipitation using Impact Protein Precipitation Plates followed by HPLC analysis. HPLC
analysis was performed using high efficiency HPLC columns using HPLC-UV, HPLC-FLD or LC/MS/MS systems.
The Impact plate allows for the analysis of 96 samples at
once, eliminates the transfer steps that are commonly associated with protein precipitation, and can also be automated. Protein precipitation was performed within the
wells of the Impact plate and sample was not allowed to
pass through the filter of the plate until vacuum was applied. This ensured that the precipitated protein was left
within the wells of the Impact plate while protein-free sample was allowed to pass through the filter and into a collection plate (Figure 1). Impact technology offers easy, fast
protein removal while providing maximized recovery of the
target analytes. The HPLC columns used produced excellent chromatographic resolution, sensitivity, and high peak
capacities for the analytes.
Figure 1. Protein precipitation using Impact Protein Precipitation Plates
Proteins
Target Analyte
Materials and Methods
Vitamin C
Protein Precipitation:
Step
1.
Place the Impact plate on top of a 96-well collection plate
2.
Dispense 300 µL of cold 5 % Meta-phosphoric acid (4 °C) into each well of the Impact plate
3.
Add 100 µL of plasma/serum samples to each well of the Impact plate
4.
Mix 5 times by aspirating with the same pipette tip
5.
Centrifuge* the Impact plate at 500 g for 5 minutes at 4 °C. Purified filtrate is collected in the
96-well collection plate.
* A vacuum manifold may be used however 25” Hg vacuum pressure must be applied for up to 10 minutes or until sample is
completely pulled through the Impact plate.
After filtrate is collected, the collection plate containing the purified samples should be covered using a sealing mat. Filtrate was transferred to a Verex™ 9 mm screw-top amber autosampler vial to protect from light
and was placed on a cooled autosampler (4 °C). The sample is now ready to be immediately injected onto
the LC/UV.
HPLC Conditions:
Gradient:
Kinetex® 5 µm XB-C18, 100 Å
150 x 4.6 mm
00F-4605-E0
SecurityGuard™ ULTRA UHPLC C18
Cartridges
Cartridge Part No.: AJ0-8768
Mobile Phase: A: 0.1 % Formic acid in water
B: Acetonitrile
Flow Rate:
Temperature:
Detection:
Injection:
Column:
Dimensions:
Part No.:
Guard:
Time (min) % B
0
0
3.5
0
3.6
100
5.0
100
5.1
0
7.0
0
800 µL/min
Ambient
UV @ 245 nm
30 µL
Vitamin B3 (Nicotinic acid and Nicotinamide)
Protein Precipitation:
Step
1.
Place the Impact plate onto a suitable 96-well sample manifold
2.
Dispense 300 µL acetonitrile into each well of the Impact plate
3.
Add 100 µL of plasma/serum samples to each well of the Impact plate
4.
Mix 3 times by aspirating with a pipette tip
5.
Apply vacuum to filter the sample and collect the purified filtrate
After filtrate is collected, the collection plate containing the purified samples should be covered using a sealing mat. The sample is now ready to be immediately injected onto the LC/MS/MS.
Materials and Methods (cont’d)
HPLC Conditions:
Column:
Dimensions:
Part No.:
Mobile Phase:
Gemini® 3 µm C18
100 x 4.6 mm
00D-4439-E0
A: 0.1 % Formic acid in water
B: Methanol
Flow Rate: 600 µL/min
Gradient: Time (min) % B
0
10
2.5
90
2.6
10
4
10
Detection: API 4000™ MS/MS, ESI Positive (ESI+)
Temperature: Ambient
Injection: 2 µL purified plasma
MS/MS Conditions:
An AB SCIEX API 4000™ triple-quadrupole tandem mass spectrometer is used for analysis
equipped with an ESI probe operating in positive polarity mode. Under MRM mode, two channels
were monitored for nicotinamide and nicotinic acid.
MRM Channel
Peak Name
Nicotinamide
123.006 ⇒ 80.100
Nicotinic acid
123.981 ⇒ 80.100
Vitamin B1 (TMP and Thiamine)
Protein Precipitation:
Step
1.
Place the Impact plate onto a suitable 96-well sample manifold
2.
Dispense 100 µL of plasma into each well of the Impact plate
3.
Add 400 µL of methanol to each well of the Impact plate
4.
Mix 3 times by aspirating with a pipette tip
5.
Apply vacuum to filter the sample and collect the purified filtrate
Transfer 50 µL to an autosampler vial (or collection plate). Add 50 µL of water and 50 µL of derivatizing
reagent (15 % of sodium hydroxide solution plus 200 µL of 30 mM K3Fe(CN)6). Cover the vial or collection
plate with a lid or sealing mat, respectively. Vortex for 15 seconds. Put the vial or collection plate into an
autosampler. Sample is ready to be injected onto the HPLC-FLD.
HPLC Conditions:
Gradient: Time (min) % B
Gemini NX-C18 3 µm
0.0
3
100 x 3.0 mm
0.25
25
00D-4453-Y0
A: 25 mM Na2HPO4, 10 % Methanol (pH 7.0)
0.75
25
B: 25 mM Na2HPO4, 70 % Methanol (pH 7.0)
3.00 35
Flow Rate: 750 µL
4.00 100
5.00 100
5.10 3
8.00 3
Temperature: 25 °C
Detection: Fluorescence (Excitation: 375 nm, Emission: 435 nm)
Injection: 20 µL
Column:
Dimensions:
Part No.:
Mobile Phase:
Figure 2. Comparison of human plasma Vitamin
C levels from extractions using 10 %
Meta-phosphoric acid vs. 5 %
Meta-phosphoric acid as a
precipitation solvent
120
10 % meta-PA
5 % meta-PA
100
60
40
App ID 21447
Peak area
80
20
0
Plasma-1
Plasma-2
Plasma-3
Figure 3. Representative standard curve of
Vitamin C at a concentration range of
0 to 100 µg/mL
Vitamin C in Human Plasma by Impact Plate
(Kinetex 5 µm, XB-C18, 150 x 4.6 mm + Guard)
3000
y = 25.567x - 6.0485
R2 = 0.9999
2500
Peak area
2000
1500
1000
500
0
0
-500
20
40
60
80
Vitamin C Concentration (µg/mL)
100
120
Table 1. Recovery of Vitamin C from spiked
human plasma
Added Vitamin C
(µg/mL)
Observed
(µg/mL)
Recovery
(%)
0
5.0
-
3
7.9
98.8
5
9.0
90.0
10
14.1
94.0
20
24.8
99.2
30
35.1
100.3
40
46.8
104.0
60
65.5
100.8
Mean
98.2
CV
4.8
Figure 4. Representative standard curve of
nicotinic acid at a concentration range
of 2 to 2000 ng/mL
20910
1.5e6
1.4e6
R2 = 0.9996
1.3e6
1.2e6
1.1e6
1.0e6
8.0e5
7.0e5
6.0e5
5.0e5
4.0e5
3.0e5
App ID 20910
Area, counts
9.0e5
2.0e5
1.0e5
0.0
0
100
200
300
400
500
600
700
800
900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 min
Concentration, ng/mL
Table 2. Signal-to-noise ratio of nicotinamide and
nicotinic acid at LOD and LOQ
Analyte
LOD
LOQ
ng/mL
S/N Ratio
ng/mL
S/N Ratio
Nicotinamide
2
4.3
10
12.4
Nicotinic acid
2
3.3
10
10.1
Table 3. Recovery of TMP and Thiamine from
human plasma after cleanup with
Impact plates
Recovery for Human Plasma - TMP
Added TMP
(nmoL/L)
Observed
(nmoL/L)
0
3.0
5
7.0
10
Recovery for Human Plasma - Thiamine
Recovery
(%)
Added Thiamine
(nmoL/L)
Observed
(nmoL/L)
Recovery
(%)
0
5.0
87.5
5
10.8
108.0
12.8
98.5
10
18.4
122.7
Mean
93.0
Mean
115.4
Table 4. Accuracy studies for TMP and Thiamine
from human plasma after cleanup with
Impact plates
TMP-Spiked Plasma Controls
Thiamine-Spiked Plasma Controls
Expected
(nmoL/L)
Results
(nmoL/L)
Accuracy
(%)
Expected
(nmoL/L)
Results
(nmoL/L)
Accuracy
(%)
1
40
38.7
96.8
40
34.2
85.5
2
20
20.0
99.8
20
19.9
99.4
3
10
10.7
107.0
10
8.2
81.8
Mean
101.2
Mean
88.9
Control No.
Results and Discussion
When developing a sample preparation procedure for vitamins and their metabolites in a high-throughput environment,
it is important to choose an effective yet rapid method. Protein precipitation was chosen to prepare our plasma samples
because it was rapid and could be automated using Impact
Protein Precipitation Plates.
Vitamin C
Vitamin C is a very polar compound that is unstable at room
temperature. Because of the instability of vitamin C we chose
to screen various precipitating solvents in order to determine
the most effective solvent for this work. It was determined
that 5 % Meta-phosphoric acid yielded the highest recoveries of vitamin C as compared to other extraction solvents
(Figure 2). Using the Impact plate to perform the precipitation step reduced the amount of time that our sample was
exposed to room temperature as compared to processing
by hand.
After precipitation, vitamin C was analyzed by HPLC-UV. Vitamin C is a very polar compound and is difficult to retain via
reversed phase HPLC; however, a Kinetex core-shell XB-C18
HPLC/UHPLC column provided a retention time of approximately 2.7 minutes, allowing us to accurately quantitate the
target analyte. Using a wavelength of 245 nm, our analysis
resulted in a correlation coefficient of 0.9999 and average
recoveries of 98.2 (Figure 3 and Table 1). A lower limit of
detection (LOD) of 0.78 µg/mL and lower limit of quantitation
(LOQ) of 1.56 µg/mL showed a signal-to-noise (S/N) ratio of
14.6 and an average S/N ratio of 14.21.
Vitamin B3 (Nicotinic acid and Nicotinamide)
Vitamin B3 exists as niacin (or nicotinic acid) as well as nicotinamide (a derivative of niacin), both of which are absorbed
from the normal diet. Nicotinic acid is a light sensitive com-
pound, making it important to ensure that it is prepared and
handled with care. When analyzing plasma samples for nicotinic acid and nicotinamide, Impact plates were used because they allowed for high-throughput analysis, provided
consistent recoveries, and reduced the amount of time spent
performing the precipitation, which helped to keep the nicotinic acid stable.
After protein precipitation, samples were immediately analyzed by LC/MS/MS using a Gemini 3 µm C18 HPLC column. The combined protein precipitation and LC/MS/
MS analysis resulted in a correlation coefficient of 0.9996
(Figure 4) and a signal-to-noise ratio of 4.3 and 3.3 at an
LOD of 2 ng/mL and 12.4 and 10.1 at an LOQ of 10 ng/mL
(Table 2).
Vitamin B1 (TMP and Thiamine)
Vitamin B1, also known as thiamine, is phosphorylated into
thiamine monophosphate (TMP) in the mucosal cells of the
duodenum. When analyzing human plasma samples for TMP
and thiamine, Impact plates were used to perform a methanol precipitation. Using the plates provided rapid cleanup,
saved time, and allowed this method to be easily automated
for increased throughput.
Following a protein precipitation step, the sample was derivatized and analyzed by HPLC-FLD using a Gemini NXC18 3 µm HPLC column. Resulting recoveries of both
target compounds averaged 93 % for TMP and 115 % for
thiamine (Table 3). Accuracy studies resulted in an average accuracy of 101.2 for TMP and 88.9 for thiamine
(Table 4), suggesting that our method not only provided acceptable recoveries but was also accurate and reproducible.
Conclusion
Vitamins are becoming a popular means to diagnose and treat
disease states. With vitamin testing on the rise, laboratories
must develop rapid, reliable, and robust analytical methods
that can be easily incorporated into a high-throughput setting. Protein precipitation has always been a popular sample
preparation method; however, the process can be improved
upon. Using Impact Protein Precipitation Plates, several different vitamins and their metabolites were cleaned up from
plasma using various precipitation solvents. The methods
can also be automated, allowing laboratories to save time
by increasing productivity while improving reproducibility and
reducing the risk of analyte instability due to extended exposure to light or high temperatures. Preparation by automated
protein precipitation is also a versatile sample preparation
technique as the resulting extract can be analyzed by several different methods including HPLC/UV, LC/MS/MS, and
HPLC-FLD.
Trademarks
Kinetex and Gemini are registered trademarks, Impact, SecurityGuard, and Verex are trademarks of Phenomenex.
API 4000 is a trademark of AB SCIEX Pte. Ltd. AB SCIEX ™ is being used under license.
Gemini is patented by Phenomenex, U.S. Patent No. 7,563,367
© 2013 Phenomenex, Inc. All rights reserved.