AN814 Vitamin D from Serum.indd

Application Note AN814
A High-Throughput method for Extraction of Vitamin B3 ISOLUTE® SLE+ | Page 1
A High-Throughput SPE Method for Extraction of
Vitamin B3 (Niacin) and Related Metabolites from
Serum Using ISOLUTE SCX-3 Prior to LC-MS/MS
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Frank Kero Ph.D., Analytical Applications Chemist, Biotage
This application note describes the extraction of nicotinic acid (niacin), nicotinuric acid and nicotinamide from
human plasma using ISOLUTE SCX-3 in 96-well plate format.
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Introduction
There are 8 water soluble analogues of vitamin B that are
relevant to the metabolic health of the human host. Of
particular interest to this study is the determination of
vitamin B3, a precursor to the synthesis of hormones that
cascade through a number of biochemical systems in vivo.
The development data presented in this report details sample
preparation workflow strategies to facilitate population
screening of the parent compound niacin (nicotinic acid, pk a s
= 2.2, 4.8) as well as 2 relevant metabolites niacinamide
(pk a = 3.54) and nicotinuric acid (pk a s = 3.1, 3.5) in a single
analysis. The structure of these analytes are shown in
Figure 1. The analytes of interest were fortified into pooled
mixed gender serum, and samples were extracted using a
25 mg format 96-well plate packed with ISOLUTE SCX-3, a
silica-based, Strong Cation eXchange sorbent (see Figure 2).
The reconstituted extracts were analysed using a gradient
mixed-mode LC-MS/MS method.
Niacinamide
Nicotinuric acid
Niacin (nicotinic acid)
Figure 1. Structures of Vitamin B3 and metabolites
Analytes
Niacin (Nicotinic acid) , nicotinuric acid, niacinamide
Sample Preparation Procedure
Format:
ISOLUTE SCX-3 25 mg plate, part number 533-0025-P01
The SPE sorbent chemistry is a ethylbenzene sulfonic acid
functionalized silica (non-end capped).
Figure 2. ISOLUTE® SCX-3 sorbent chemistry
Sample Pre-treatment: Dilute serum (50 µL) with aqueous acetic acid (2%, 150 µL). Mix thoroughly.
Conditioning:
Condition each well with methanol (1 mL)
Equilibration:
Equilibrate each well with aqueous acetic acid (2%, 1 mL)
Sample Loading:
Load pre-treated sample (200 µL) at a flow rate of 1 mL/min using positive pressure
(PRESSURE+96 Positive Pressure manifold PPM-96)
Interference Wash 1:
Wash each well with water:methanol:acetic acid (68:30:2, v/v/v, 2 x 1 mL)
Interference Wash 2:
Wash each well with methanol:acetic acid (98: 2, v/v, 2 x 1 mL)
Analyte Elution:
Elute analytes with methanol: ammonium hydroxide (95:5, v/v, 2 x 400 µL)
Post Extraction:
Evaporate extracts to dryness and reconsitute in 0.1% formic acid (100 µL)
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Application Note AN814 ©2014 Biotage
A High-Throughput method for Extraction of Vitamin B3 ISOLUTE® SLE+ | Page 2
HPLC Conditions
Instrument:
Agilent 1200 Liquid Handling System (Agilent Technologies, Berkshire, UK)
Column:
IMTAKT Scherzo SM-C18 column (2 mm x 150mm, 3.0 µm) (IMTAKT USA,
Philadelphia, USA)
Injection volume:
20 µL
Mobile Phase:
Solvent A: 5mM ammonium formate / 0.1% FA (aq)
Solvent B: Acetonitrile
Gradient:
Table 1. Gradient parameters for the sepration of vitamin B3 and related metabolites
Step
Time (min)
Flow Rate
(µL/min)
%A
%B
1
0.5
200
80
20
2
3
3.0
200
70
30
4.0
200
70
30
4
5.0
200
80
20
5
8.0
200
80
20
Mass Spectrometry Conditions
Instrument:
Applied Biosystems /MDS Sciex 4000 Q-Trap hybrid triple quadrapole /
linear ion trap mass spectrometer (Applied Biosystems, Foster City, CA.)
equipped with a Turbo Ionspray interface operated in positive ion mode.
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Ion Source Temperature:
600 oC
The MRM transitions used in this study were detailed in Table 2. Data acquisition can stop collecting
after 4.5 min. (to facilitate multiplexed column switching methods).
Table 2. MS/MS transitions for the detection of vitamin B3 and related metabolites
Analyte
Niacin
g/mole
MRM Transition
(m/z)
Declustering
Potential
(DP)
Collision
Energy
Dwell Time
(ms)
80.1
30
27
16
123.1
124.1
Niacinamide
122.1
123.1
80.0
30
25
16
Nicotinuric acid
180.0
181.0
79.0
30
28
16
Reagents
HPLC grade water, methanol, acetonitrile, nicotinuric acid, ammonium hydroxide, acetic acid and formic acid (FA)
were purchased from Sigma-Aldrich Co. (Atlanta, GA.). Nicotinic acid and niacinamide standards were obtained
from Cerilliant Corp (Round Rock, TX). The biological fluids were obtained from BioChemEd Services.
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Application Note AN814 ©2014 Biotage
A High-Throughput method for Extraction of Vitamin B3 ISOLUTE® SLE+ | Page 3
Results and Discussion
The sorbent selection for this study proved interesting as nicotinic acid and nicotinuric acid have both acid and
basic pK a values. A series of polymer-based cation exchange and anion exchange sorbents were evaluated;
however, the ethylbenzene sulfonic acid functionalized silica demonstrated the best choice, inclusive of all
three analytes.
A representative chromatogram for a 20 ng/mL fortified serum sample is shown in Figure 3. A set of fortified
serum specimens was also prepared at 40 ng/mL levels (n=6). The relative recovery plot is detailed in Figure 4.
The method repeatability as %RSD was determined <15% for all analytes.
Optimization of the method conditioning step was conducted by testing the following solutions: 2% formic acid,
2% acetic acid, 4% acetic acid, 50 mM NH4 Ac (pH=6) and 50 mM NH4 Ac (pH=7). The peak area response for 2%
acetic acid provided the best results, inclusive of all three analytes.
A gradient mix of water and methanol was evaluated for analyte response. It was determined that incorporating
2% acetic acid into the optimized 70/30 water/MeOH solution was helpful in maintaining analyte recoveries.
Acetonitrile did not offer any advantage as wash solvent or an elution solvent. Evaluation of the second wash
solvent did not show analyte response as breakthrough in the wash. There was a significant benefit in the
quality of the data obtained when increasing the wash volumes in replicate aliquots. The analyte suppression
values were observed at < 20% (Figure 5). A loss of analyte recovery was observed after 3 wash cycles so a
balance of analyte recovery vs sample cleanliness should be considered when targeting clinically relevant LOQ
values.
Alternative solvents for elution were considered in an effort to maintain adequate relative recovery for all of the
analytes. The utility of ethyl acetate and ammonium hydroxide has been demonstrated in mixed-mode cationexchange applications. To mitigate compatibility issues with ethyl acetate and water, a drying step was added
after the wash 2 step. In this study, this combination of solvents eluted cloudy and was therefore excluded from
consideration.
The ISOLUTE SCX-3 96-well plate format demonstrated as a viable option for serum measurements over a
relevant concentration range in clinical diagnostics.
Figure 3. A typical chromatogram obtained from the extraction of a 20 ng/mL fortified
specimen of plasma
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Application Note AN814 ©2014 Biotage
% Suppression
Relative recovery (%), n=6
A High-Throughput method for Extraction of Vitamin B3 ISOLUTE® SLE+ | Page 4
nicotinic acid
niacinamide
Repeat wash cycles
nicotinuric acid
nicotinic acid
Figure 4. Relative recovery (%) and method repeatability for the
extraction of vitamin B3 and related metabolites from serum.
niacinamide
nicotinuric acid
Figure 5. Matrix suppression determined from the extraction of
vitamin B3 and related metabolites from serum.
Acknowledgment:
Biotage would like to thank IMTAKT USA (1315 Walnut Street, Suite 619, Philadelphia, PA 19107 http://www.imtaktusa.com/)
for providing the HPLC column for this study.
Ordering Information
Part Number
Description
533-0025-P01
ISOLUTE -96 SCX-3 25 mg plate
Quantity
1
SD-9600-DHS-EU
Biotage SPE Dry Sample Concentrator System 220/240 V
1
SD-9600-DHS-NA
Biotage SPE Dry Sample Concentrator System 100/120 V
1
PPM-96
Biotage Positive Pressure Manifold 96 Position
1
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Part Number: AN814
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