The document discusses the development of a high-throughput LC-MS/MS method for the simultaneous quantification of all-trans retinol and alpha-tocopherol in human serum, demonstrating high correlation to reference methods. It highlights the assay's precision, linearity, and recovery while noting the lack of interference from substances like bilirubin and triglycerides, with hemolysis being a rejecting factor. The method's stability and performance characteristics were thoroughly evaluated, establishing its reliability for clinical applications.

1. Specimen Stability
The LC-MS/MS methods demonstrate a high degree of correlation to the reference methods.
Accuracy
Measurement of All-Trans Retinol and Alpha-Tocopherol in Human
Serum by Liquid Chromatography-Tandem Mass Spectrometry
Alpha-Tocopherol Linearity
y = 0.9826x + 0.4294
R
2
= 0.9967
0.0
6.0
12.0
18.0
24.0
30.0
0.0 6.0 12.0 18.0 24.0 30.0
Expected
Measured
Ascorbic Acid Lithium Heparin Comparison
y = 0.9964x + 0.1296
R2
= 0.9936
0.0
5.0
10.0
15.0
20.0
25.0
0.0 5.0 10.0 15.0 20.0 25.0
BGL HPLC
MMLNELC-MS/MS
y = 1.0942x + 0.1552
0
5
10
15
20
25
30
35
0 5 10 15 20 25 30
BGL HPLC
MMLNELC-MS/MS
D.M Garby1, K. Kloke2, P.M. Ladwig2, R. DelRosso1, L.A. Cheryk1
1Mayo Medical Laboratories, Andover, MA, 2Mayo Clinic, Rochester, MN
Precision
• The effect of interfering substances was assessed by spiking samples of known
concentrations of all-trans retinol or alpha-tocopherol with various substances
(hemoglobin, bilirubin and triglyceride (Intralipid)) and then comparing the spiked
sample result to that of the non-spiked sample result. The presence of bilirubin or
triglycerides did not demonstrate interference in the assays. Specimens that are
hemolyzed should be rejected. Lipemia demonstrates some degree of interference in
the alpha-tocopherol assay.
• No ion suppression was detected during post-column infusion of the internal standards.
Ion suppression was also assessed by spiking internal standard into a serum sample and
then performing serial dilutions with deionized water. Linear regression of the
expected versus observed internal standard area, expressed in counts per seconds,
would demonstrate a non-linear response in the presence of ion suppression. Linear
regression of the data yielded the following results for all-trans retinol-d5: y = 1.022x
and a correlation coefficient, R = 0.9975. Linear regression of the data yielded the
following results for alpha-tocopherol-d6: y = 0.9553x and a correlation coefficient,
R = 0.9965. No ion suppression was demonstrated.
Interferences and Ion Suppression
• All-trans retinol and alpha-tocopherol are essential vitamins that play a role in various
cellular activities. Quantification of these vitamins can provide important clinical
information for the assessment of vitamin deficiency or excess.
• We have developed a high throughput LC-MS/MS assay for the determination of all-
trans retinol and alpha-tocopherol levels simultaneously in serum that offers the
advantages of improved specificity and shorter analytical time.
Conclusions
1. Bushue N, Wan Y-JY. Retinoid pathway and cancer therapeutics. Adv Drug Deliv Rev
2010;62:1285-1298.
2. Shenkin A, Baines M, Fell GS, Lyon TDG. Vitamins and trace elements. In: Burtis CA, Ashwood
ER, Bruns DE, eds. Tietz textbook of clinical chemistry and molecular diagnostics. 4th Ed. St. Louis:
Elsevier Saunders 2006:1079-1087.
3. Duriancik DM, Lackey DE, Hoag KA. Vitamin A as a regulator of antigen presenting cells. J Nutr
2010;140:1395-1399.
References
Background: Vitamin A (retinol) is a fat-soluble vitamin that plays an essential
role in the function of the retina (adaptation to dim light), is necessary for growth and
differentiation of epithelial tissue, and is required for growth of bone, reproduction,
and embryonic development.1,2 Vitamin A must be obtained through the diet and
includes retinol, retinyl ester and beta-carotene.1 Deficiency of vitamin A has
profound negative effects on the immune response leading to an increased
susceptibility to infection.3 Vitamin E (alpha-tocopherol) is a fat-soluble vitamin that
contributes to the normal maintenance of biomembranes, along with its requirement
for proper neurological and reproductive function as well as its role as an antioxidant
and free-radical scavenger protecting the integrity of unsaturated lipids in the
biomembranes of all cells.2 Vitamin E is commonly obtained from dietary oils and
fats such as wheat germ oil, sunflower oil, grains and nuts.2
Methodology: Deuterated stable isotopes (all-trans retinol-d5 or alpha-
tocopherol-d6) were added to 50 uL of human serum as internal standards. Protein
was then precipitated from the mixture by the addition of acetonitrile. All-trans
retinol, alpha-tocopherol and the internal standards were extracted via an on-line
extraction utilizing high-throughput liquid chromatography (TLX4, Thermo Fisher
Scientific, Waltham, MA) followed by conventional liquid chromatography and
analysis on a tandem mass spectrometer (API 5000, AB Sciex, Foster City, CA)
equipped with a heated nebulizer ion source. Ion transitions monitored in the
multiple reaction monitoring (MRM) mode were m/z 269.3 → m/z 93.1 for all-trans
retinol, m/z 274.4 → m/z 93.1 for all-trans retinol-d5, m/z 430.4 → m/z 165.2 for
alpha-tocopherol and m/z 436.4 → m/z 171.3 for alpha-tocopherol-d6. Calibrators
consisted of six standard solutions ranging from 0 to 200 ug/dL for all-trans retinol
and 0 to 30 mg/L for alpha-tocopherol.
Results: Method performance was assessed using precision, linearity, recovery
and specimen stability. Precision studies were performed using National Institute of
Standards (NIST) standard reference material (SRM), and bovine serum spiked with
all-trans retinol and alpha-tocopherol standard solutions. Intra-run precision
coefficients of variation (CVs) ranged from 1.0% to 1.3% for all-trans retinol and
1.1% to 2.3% for alpha-tocopherol. All-trans retinol inter-run precision CVs ranged
from 3.0% to 6.5%, and alpha-tocopherol inter-run precision CVs ranged from 2.0%
to 8.2%. Linearity studies were performed using bovine serum spiked with all-trans
retinol and alpha-tocopherol standard solutions. Linearity was demonstrated over
each assay range (2 to 200 ug/dL for all-trans retinol and 0.5 to 30 mg/L for alpha-
tocopherol) yielding the following equations: observed all-trans retinol value =
0.9824*(expected value), R2 = 0.9993; observed alpha-tocopherol value =
0.9826*(expected value) + 0.4294, R2 = 0.9967. Recovery averaged 101% for all-
trans retinol and 103% for alpha-tocopherol across the assay range. A stability study
demonstrated that specimens are stable at ambient, refrigerate and frozen (-20ºC or
lower) temperatures for up to 14 days.
Conclusions: This method provides for the simultaneous and reliable high
throughput analysis of all-trans retinol and alpha-tocopherol in serum.
Abstract
Interference Studies: The potential for method interference from hemoglobin,
bilirubin, and lipemia was assessed by spiking known concentrations of these possible
interferences into bovine serum fortified with all-trans retinol or alpha-tocopherol
standard solutions and comparing the data to the results obtained in the absence of
interferences.
Experimental Design
 2011 Mayo Foundation for Medical Education and Research
Deuterated stable isotope (all-trans retinol-d5 or alpha-tocopherol-d6) was added to 50
uL of serum as an internal standard. Protein was then precipitated from the mixture by
the addition of acetonitrile. All-trans retinol, alpha-tocopherol and the internal
standards were then extracted from the resulting supernatant via an on-line extraction
utilizing high-throughput liquid chromatography (HTLC) followed by conventional
liquid chromatography (LC).
The mobile phases for the on-line extraction consisted of an aqueous phase (98/2 v/v,
deionized water/acetonitrile, 0.005% formic acid), organic phase (methanol, 0.005%
formic acid) and a cleaning solvent (50/50 v/v, acetone/isopropyl alcohol). An HTLC
C18 column 0.5mm x 50mm (Thermo Scientific, Santa Clara, CA) was used for the
on-line extraction. The mobile phases for the analytical chromatography consisted of
an aqueous phase (98/2 v/v, deionized water/acetonitrile, 0.005% formic acid) and an
organic phase (50/50 v/v, acetonitrile/methanol, 0.1% formic acid). A Kinetex C18
100mm x 4.6mm, 2.6µm (Phenomenex, Torrance, CA) was used as the analytical
column. The LC-method was multiplexed utilizing a TLX-4 chromatography system.
Final analysis was performed using a tandem mass spectrometer (API 5000) equipped
with a heated nebulizer ion source. The operating conditions are as follows: source
temperature of 450°C, nebulizer gas (GS1) at 40 and curtain gas at 20. Ion transitions
monitored in the multiple reaction monitoring (MRM) mode were m/z 269.3 → m/z
93.1 for all-trans retinol, m/z 274.4 → m/z 93.1 for all-trans retinol-d5, m/z 430.4 →
m/z 165.2 for alpha-tocopherol and m/z 436.4 → m/z 171.3 for alpha-tocopherol-d6.
Calibrators consisted of six standard solutions ranging from 0 to 200 ug/dL for all-trans
retinol and 0 to 30 mg/L for alpha-tocopherol.
Assay Principle Results
All-Trans Retinol Intra-assay
(N=20)
All-Trans Retinol Inter-assay
(N=15)
Mean ± SD
(ug/dL)
%CV Mean ± SD
(ug/dL)
%CV
14.6 ± 0.2 1.3 14.2 ± 0.6 4.1
29.3 ± 0.3 1.1 28.5 ± 1.6 5.8
103.7 ± 1.2 1.1 101.2 ± 5.0 5.0*
Alpha-Tocopherol Intra-assay
(N=20)
Alpha-Tocopherol Inter-assay
(N=17*, N=20)
Mean ± SD
(mg/L)
%CV Mean ± SD
(mg/L)
%CV
2.1 ± 0.1 2.3 2.2 ± 0.1 5.6*
5.4 ± 0.1 1.3 5.7 ± 0.3 5.7
27.2 ± 0.2 0.9 27.7 ± 1.0 3.7
Linearity
Alpha-Tocopherol Stability – Storage in Amber Tubes
Day 1
(% change)
Day 3
(% change)
Day 7
(% change)
Day 14
(% change)
20-25°C -6.3 -1.6 5.0 -8.1
2-8°C -5.7 1.4 7.3 4.9
-20°C -7.9 -0.7 3.8 -0.3
Effect of Freeze-Thaw Cycles
1 cycle 2 cycles 3 cycles 4 cycles
% change -1.9 -1.3 7.6 -4.3
All-trans retinol demonstrated comparable stability data when stored under the same conditions.
Precision: Intra-assay and inter-assay precision for all-trans retinol and alpha-
tocopherol was demonstrated using commercially available quality control material
and bovine serum spiked with all-trans retinol or alpha-tocopherol standard solutions.
Intra-assay precision was determined from 20 serial measurements of each sample.
Inter-assay precision was calculated from 15, 17 or 20 replicate determinations of each
sample. The mean, coefficient of variation (CV) and standard deviation (SD) were
calculated at each level for each method.
Accuracy: The new LC-MS/MS method was compared to the existing HPLC
method (N=48) performed at the Mayo Clinic, Biochemical Genetics Laboratory,
Rochester, MN.
Linearity: Linearity covering the all-trans retinol and alpha-tocopherol reportable
ranges was assessed by performing serial dilutions of bovine serum spiked with all-
trans retinol or alpha-tocopherol standard solutions. Each level was run in triplicate
and plotted versus the expected concentrations.
Specimen Stability: Specimen stability was assessed under ambient, refrigerate
and frozen (-20ºC) storage conditions and the effect of multiple freeze thaw cycles was
also evaluated.
Ion Suppression: Ion suppression was assessed by post column infusion of all-
trans retinol-d5 or alpha-tocopherol-d6 and data was collected at the expected
retention time of all-trans retinol or alpha-tocopherol. Suppression of signal is
visualized by the presence of an inverse peak. A second method to detect ion
suppression was also utilized. A serum sample was spiked with all-trans retinol-d5 or
alpha-tocopherol-d6 and then serially diluted using deionized water. Each level was
run in triplicate and plotted versus the expected counts per second. Ion suppression
would be detected with this technique via a non-linear regression line.
Experimental Design
y = 0.9864x + 4.0003
0
200
400
600
800
1000
1200
1400
1600
0 500 1000 1500
BGL HPLC
MMLNELC-MS/MS
All-Trans Retinol Comparison
Alpha-Tocopherol Comparison
R = 0.98
R = 0.95
All-Trans Retinol Linearity
y = 0.9824x
R
2
= 0.9993
0.0
50.0
100.0
150.0
200.0
0.0 50.0 100.0 150.0 200.0
Expected
Measured