Quantitative retinol assay for serum and dried blood spots

Capillary Zone Electrophoresis (CZE) with laser-excited fluorescence detection has been found to be a fast, easy, and accurate method for directly measuring serum retinol. Due to its small sample requirements, the method may be used for finger-prick analysis.

BACKGROUND OF THE INVENTION 
As is well-known, vitamin A is extremely important for the health of humans 
and animals. Among its essential functions are cellular differentiation 
and vision. Deficiency of Vitamin A can result in adverse effects on 
reproduction, growth, and the immune response. Vitamin A deficiency can 
also affect the eyes often resulting in blindness. 
On the other hand, high levels of vitamin A can cause serious toxic 
manifestations; namely teratogenicity, chronic toxicity and acute 
hypervitaminosis. Some individuals may also show a genetic sensitivity to 
vitamin A, termed vitamin A intolerance, at intakes not much above those 
normally ingested. Thus, management of Vitamin A levels can have serious 
implications. 
Presently in Third World nations, many young children are plagued by night 
blindness and other vitamin A-related diseases. It has been estimated that 
more than one million become permanently blind each year because of 
vitamin A deficiency. 
Accordingly, for the above reasons, development of a fast and accurate 
method of measuring vitamin A levels is of international importance. This 
invention relates to a method of determining vitamin A levels in human 
blood serum which offers the advantage of smaller blood samples for 
analysis as well as less time and more simplicity. 
Vitamin A (retinol) is normally transported in the blood as a complex with 
RBP (retinol-binding protein). RBP is a single polypeptide chain with a 
molecular mass of about 21,000 and has a single binding site for one 
molecule of retinol. The retinol-RBP further interacts strongly with 
another protein, plasma transthyretin (TTR or prealbumin) and normally 
circulates in plasma as a 1:1 complex (molar ratio) with TTR. These 
complex interactions and association of companion proteins with retinol 
have made determining retinol levels a difficult, time-consuming task. 
The serum retinol concentration is the most commonly used indicator of 
vitamin A status. The preferred method for analysis of retinol is 
high-performance liquid chromatography (HPLC). The method involves 
collection of at least 200 .mu.l of whole blood, centrifugation within 
hours of collection, and keeping the samples in the cold during transport 
and storage. In the laboratory, the serum proteins are precipitated and 
retinol is extracted with organic solvents. The extracts are separated by 
HPLC and retinol is detected by UV absorbance or fluorescence. The 
limitations of this method are: (1) a large amount of serum sample (100 
.mu.l or more) is needed, which is often difficult to obtain by capillary 
sampling and represents a large volume from infants, especially for 
neonates and low-birth-weight infants; (2) once separated from RBP, 
retinol is light-, oxygen- and heat-sensitive, increasing the likelihood 
of error during analysis; (3) processing time is relatively long. 
Retinol is labile to light, oxygen, and heat, making it difficult to 
handle, especially when it is removed from the protection of its 
biological matrices. Extracted retinol decomposes rapidly even at 
subambient temperature when exposed to normal light. 
In contrast, retinol is stable in frozen serum at -70.degree. C. for at 
least eight years. Retinol itself has a maximum absorbency at 325 nm and 
fluorescences at 425 nm. However, when retinol is bound to RBP the 
intensity of the fluorescence is enhanced ten to fourteen-fold and the 
fluorescence shifts to 465 nm. Additionally, these characteristics of 
retinol have provided encouragement for methods that would allow a direct 
determination of retinol in serum. 
Current improvements along this vein include a micromethod involving 
gel-electrophoretic separation of serum, with subsequent estimation of the 
retinol-RBP complex by fluorimetric scanning of the gel. This method 
avoids solvent extraction, but is still limited by large sample 
requirements, long separation times and gel scanning, which make it of 
limited use for surveys of vitamin A status. 
Recently, high performance size-exclusion liquid chromatography (SE-HPLC) 
with fluorescence detection has been used to measure retinol-RBP in animal 
and human serum. Compared to previous methods, less serum is required and 
preliminary sample treatment is avoided. However, a 0.05 ml blood sample 
is needed for analysis and 20-30 minutes of HPLC separation time is 
required. In addition, at least 1 ng of retinol-RBP is required for 
detection. These factors make it difficult to do minimicroassy, especially 
for babies and young children, where only small blood samples are 
available. 
There is therefore a need in the art for a method to directly measure 
retinol in blood serum quickly, accurately, and without need for large 
blood samples. 
The primary object of the present invention is, therefore, to develop a 
method to directly evaluate levels of vitamin A in human blood serum. 
It is a further object of the present invention to provide a method to 
determine vitamin A levels in human blood serum which is accurate, fast, 
and easy. 
It is a still further object of the present invention to develop a method 
to determine blood serum levels of vitamin A which requires smaller blood 
samples by use of Capillary Zone Electrophoresis than conventional 
methods. 
SUMMARY OF THE INVENTION 
This invention relates to the use of Capillary Zone Electrophoresis (CZE) 
with fluorescence detection in separating and detecting retinol in human 
blood serum. Serum taken from a human subject is injected into a capillary 
for separation along an electrical gradient maintained within the 
capillary. A buffer maintained at a pH between 7.5 and 10.3 is used to 
carry the serum through the device. A laser or other device with emission 
at the 325 nm wave length is used for excitation and the florescence of 
the vitamin A-retinol binding protein (RBP) complex is then collected and 
measured. 
In this system, the sample size may be from 8 to 10 nl and is injected 
without any additional sample preparation to purify the retinol. The 
analysis time for each sample is less than six minutes and subfemtomoles 
of vitamin A can be easily detected. This methodology also may be used to 
measure serum retinol levels from a dried blood spot which is dissolved 
and then subjected to CZE. CZE offers many advantages over the most 
commonly used method to measure serum retinol, HPLC, which include higher 
resolution and shorter analysis time. In addition, it requires only a very 
small amount of sample (nl or pl) and has a very low detection limit 
(attomoles).

DETAILED DESCRIPTION OF THE INVENTION 
CZE does not require separation of the retinol from the serum prior to its 
analysis. Because of this, the CZE method is much less time-consuming and 
easier to use than the HPLC method. In addition, since the CZE method 
requires only a very small amount of blood sample for analysis, CZE is 
potentially useful for finger prick vitamin A analysis, especially for 
babies and young children, as well as dried blood spot analysis which will 
greatly simplify sample collection. To date no method has been developed 
using Capillary Zone Electrophoresis to detect serum retinol. 
Generally the method is as follows. First a sample of serum from a patient 
is prepared by centrifugation. The amount of blood taken can be quite 
small as only 5 to 10 uL of serum is needed. For dried blood spot analysis 
the blood is spotted onto filter paper which is dried and later dissolved 
with buffer. The sample is then pre-treated with a urea buffer or any such 
buffer which includes an agent such as urea which will denature 
transthyretin (TTR) from the TTR-Retinol-RBP complex without dissociating 
the retinol-RBP. This extra step not present in traditional protocals was 
found to be necessary because of problems with sample absorption onto the 
capillary poor separation and quantitation. 
Running buffer then is added to the serum (or dissolved serum). Suitable 
buffers include any traditional buffer solution known to those of skill in 
the art and can include borate, tris phosphate, phosphate or any agent 
which will not react with the retinol. Concentrations of the buffer can be 
from 30 to 70 mM, and must be adjusted to a pH in the range of from 
7.5-10.3 the pH range is critical for the accurate detection of retinol 
RBP. Buffer is added according to the following proportions which may vary 
depending on amount of serum. Generally to 10 uL of serum, 200-500 
preferably about 300 uL of pretreatment buffer is added. The mixture is 
filtered to remove larger molecular weight molecules (&gt;30 kDa) and then is 
ready to be injected into the capillary device with running buffer in an 
amount of 5-10 nL. 
Capillaries are those commonly used in this type of electrophoric 
separation. Traditionally they are glass or silica with diameters ranging 
from 25-70 uM. Any polymer coating on the capillary is removed towards the 
cathodic end to create a detection window. An electrical gradient is set 
up along the length of the capillary, to establish an electrophoresis 
device as is known to those of skill in the art. 
An emission source such as a laser which will emit radiation at a 
wavelength of 325 nm is used for excitation and the resulting retinol 
florescence at 465 nm is amplified for signal detection. A filter is used 
in a preferred embodiment to reject stray and scattered radiation from the 
laser head. 
A photodiode is used to convert the collected light energy at the filtered 
wavelength to an electrical signal over time which may be integrated. 
An electronic reporting integrator is used to translate the signal into a 
series of peaks defined by arbitrary units over time. Quantitation of 
florescence is achieved by comparison of peak areas or peak heights to a 
standard calibration curve, prepared by determination of detector response 
(peak height or peak area) to known amounts of retinol analyzed under 
identical conditions. Peak area is preferred because it is less 
susceptible to fluctuations due to varying conditions. 
When retinol is measured by this method a response is achieved in less than 
6 minutes and is linear over the range 0.1-0.6 .mu.g/ml, which is the 
physiological range in human serum. 
The following examples are offered to further illustrate, but not limit the 
invention. They show the comparison of HPLC versus CEZ analysis, also 
illustrate the effects that changing the pH of the buffer has on the 
retinol-RBP complex, as well as the method using a dried blood spot 
sample. 
EXAMPLE I 
Reagents: 
All chemicals are of analytical reagent grade unless stated otherwise. 
Deionized water was prepared with a Milli-Q system (Millipore, Bedford, 
Mass.). 
Running Buffer Preparation: 
The running buffer is composed of 50 mM Na.sub.2 HPO.sub.4, the pH is 
adjusted to 7.8 with 1.5M H.sub.3 PO.sub.4. The running buffer is filtered 
with 0.45 .mu.m membrane and degassed before using. 
Serum Samples: 
Actual samples of serum were also obtained. Frozen human serum was obtained 
from the Department of Nutritional Science in the University of 
Connecticut (Storrs, Conn., USA). Fresh human blood samples were obtained 
from volunteers in Grim-Smith Hospital (Kirksville, Mo., USA). All the 
blood samples were centrifuged for 7-8 minutes at 3700 rpm (approximately 
1500 g) to separate red blood cells from the serum. Serum was then 
directly injected into the high-performance capillary zone electrophoresis 
(HPCZE) for analysis. Unused serum was kept at -20.degree. C. until 
analysis. 
Pretreatment of Serum Samples: 
To 10.mu. of serum, 290.mu. of ice-cold sample pretreatment buffer (50 mM 
Na.sub.2 HPO.sub.4 +6M urea pH 7.8) was added and the mixture was then 
mixed well on a Vortex mixer (Fisher, St. Louis, Mo., USA). The 
pretreatment buffer pH had adjusted to 7.8 with 1.5M H.sub.3 PO.sub.4. The 
solution was then allowed to stand on ice for 3-5 minutes and 100 .mu.L 
solution was taken out and put into a Microcon-30 filter unit (Amicon, 
Inc., Beverly, Mass., USA) (this filter unit allows molecular weight &lt;30 
KDa molecules to pass through), and filtered by centrifugation. The 
filtrate was then ready to be injected into the high performance capillary 
electrophoresis (HPCE) column for analysis. 
Standard Retinol-RBP Preparation: 
A standard was used to calibrate the system. Standard Retinol-RBP was 
prepared using a modification of the method of the Moffa and Krause. 
First, 10 mg of pure retinol standard was dissolved in 10 ml of absolute 
ethanol (concentration was 1g/l). 10 .mu.m of standard RBP was dissolved 
in 5 ml of 0.1M sodium phosphate buffer (pH 7.7). Then 20 .mu.m of retinol 
standard solution was slowly transferred into 5 ml of standard of RBP 
solution with appropriate stirring. The retinol-RBP mixture was allowed to 
stand for 3 hours at 25.degree. C. and was then ready for injection. 
Equipment Set-Up: 
A Model CZE 1000R high-voltage power supply (Spellman, Plainview, N.Y., 
USA) was used to supply the electromotive force across the capillary. The 
anodic high-voltage end of the capillary was isolated in a plexiglass box 
for safety while the cathodic end was held at ground potential. A 60 cm 
.times.50 .mu.m I.D. fused silica capillary tubing (Polymicro Techniques, 
Phoenix, Ariz., USA) was used for the separation. The polymer coating was 
burned off 25 cm from the cathodic end of the capillary to form the 
detection window. 
A Model 4240NB helium-cadmium (He-Cd) laser (Liconix, Santa Clara, Calif., 
USA) operating at 325 nm was used for excitation. A band-pass filter (250 
nm-400 nm) (Ealing, Holliston, Mass.: Model UG-11) was used to reject 
stray and scattered radiation from the laser head. The laser was focused 
onto the capillary with a 1 cm focal length lens, and the fluorescence was 
collected with a 10x microscope objective at a 90.degree. angle to the 
incident light. The fluorescent image was focused onto a silicon 
photodiode combined with a build-in amplifier (Hamamatsu, Bridgewater, 
N.J.; Model HC220-01). Another band-pass filter (400-539 nm) (Ealing, 
Holliston, Mass.; Model 35-532) is used to isolate the fluorescence (465 
nm) from the vitamin A-RBP complex. The voltage from the photodiode was 
monitored with an autoranging microvolt DMM (Keithley, Cleveland, Ohio; 
Model 197AZ) and the signal was recorded with a Model C-R3A integrator 
(Shimadzu, Columbia, Md.). 
Pretreatment of the Capillary Column: 
Each new capillary column was filled with a 1.0M sodium hydroxide solution 
for about 30 minutes to clean the column. The column was then washed with 
a 0.1M sodium hydroxide followed by deionized water and finally running 
buffer. The capillary was ready for use thereafter. 
Conventional Extraction and HPLC Analysis of Retinol From Serum: 
Standard Extraction and HPLC was performed to compare the to methods. HPLC 
was performed as follows. To 100 .mu.l of the freshly thawed serum from 
the Department of Nutritional Science was added an equal volume of 
methanol containing an internal standard (retinyl hexanoate): each sample 
was extracted three times with an equal volume of hexane. Hexane from 
combined hexane extracts was evaporated under a gentle stream of argon, 
and the residue was dissolved in 50 .mu.l 2-propanol-dichloromethane; 20 
.mu.l was injected for HPLC analysis. Samples were analyzed by 
reversed-phase HPLC on a 5-.mu.m Resolve C.sub.18 column (Waters Assoc., 
Milford, Mass., USA) using a mobile phase of 
acetonitrile-dichloromethane-methanol-n-butanol (90:15:10:01), containing 
0.1% ammonium acetate, at a flow-rate of 1.0 ml/minute, with detection at 
300 nm. 
HPCZE Analysis: 
Serum samples were injected electrokinetically at 10 kV for 10 seconds 
(approximately 8-10 nl were injected), and the separation was carried out 
at 24 kV for 5 minutes. The electrophoretic current was monitored with a 
multimeter throughout the separation to ensure the reproducibility. After 
five to seven injections, the capillary required cleaning due to the 
adsorption of serum proteins on the capillary wall. Cleaning was 
accomplished by flushing the capillary for 4 minutes with 1M sodium 
hydroxide, then 2 minutes with deionized water and finally for 2 minutes 
with running buffer. 
Frozen human serum from the Department of Nutritional Science was used to 
make a calebration curve to quantify serum retinol levels. 
Effect of pH on Separation and Signal: 
Adsorption of proteins onto the capillary wall is a serious problem when 
separating proteins by CZE using uncoated silica columns. Performing the 
separation at a pH above the isoelectric point(p1) of the proteins under 
investigation is one of the most active ways to minimize protein buildup. 
In this way the coulombic repulsion between negatively charged proteins 
and the capillary wall will minimize the protein adsorption. However, the 
retinol-RBP complex is sensitive to the pH of the buffer, whereby higher 
pH may cause the retinol-RBP complex to decompose. 
In order to optimize the separation and detection of the retinol-RBP 
complex, a wide range of buffer pH (3-11.5) was investigated and over 15 
different pH buffers were tried before the critical pH range was 
established. At lower pH (2), no retinol-RBP signal was observed. Two 
phenomena contribute to this observation. First, since the pl of RBP is 
between 4.4 to 4.8 the retinol-RBP complex was heavily adsorbed onto the 
capillary when the pH of the buffer was near to or lower than the pl of 
the RBP. Secondly, the fluorescent intensity of the retinol-RBP complex at 
this pH is reduced. Both factors have been demonstrated experimentally. 
When the pH of the buffer was 6-7.2, only a single peak with a small 
shoulder was observed. In addition, there was no linear relationship 
between peak heights or peak areas and retinol-RBP levels for a series of 
standard samples at low or neutral pH. This may be due to incomplete 
separation of the serum matrix and retinol-RBP and the partial adsorption 
of retinol-RBP onto the capillary wall. When the buffer pH was kept at 
11.5, the characteristic peak of retinol-RBP disappeared. This was 
apparently due to the decomposition of retinol-RBP complex. If the buffer 
pH was kept at 10.3, the retinol-RBP peak was detectable: however, the 
linearity of the retinol-RBP response of the standard serum samples was 
very poor due to partial decomposition of the retinol-RBP complex. When 
the buffer pH was maintained between 7.5 and 8.5, both complete separation 
of the retinol-RBP complex from other serum components and a linear 
response for standard serum samples were attained. However, within this pH 
range, we observed that the fluorescent signal of the retinol-RBP complex 
was enhanced at higher pH (from 7.8 to 8.5). Therefore, pH 7.8 was 
employed in this work as an optimized pH to analyze serum samples. 
Identification of retinol-RBP Peak: 
In order to verify the retinol-RBP peak in the standard serum 
electropherogram, pure retinol-RBP complex, which was prepared from 
retinol and isolated human RBP was injected. The results was shown in FIG. 
3. The pure retinol-RBP complex migrates as a single peak (A) that exactly 
matches the peak A in FIG. 2. 
Although retinyl esters, as well as very small amounts of unesterified 
retinol, will be present in chylomicra, their contribution to circulating 
vitamin A in fasting blood is small (&lt;10%); retinol-RBP is the 
physiologically important transport form of vitamin A. Retinol-RBP is well 
resolved from the lipoproteins by these electrophoretic conditions. 
Linearity: 
Linear response to graded levels of vitamin A in the serum is extremely 
important in quantitative analysis of vitamin A in human serum. We 
observed very good linearity between the fluorescence signal (peak height) 
and the vitamin A concentration of standard serum samples with known 
concentrations of vitamin A. The response is linear over the range 0.1-0.6 
.mu.g/ml which is the physiological range in human serum. The detection 
limit for this technique is approximately 10 ng/ml of serum (or 10 fg of 
retinol) at a signal-to-noise of 5:1. In order to make sure the separation 
conditions are maintained the same, a fresh serum sample was used as a 
reference, which was injected after several sample injections. 
Comparison of HPCZE with HPLC for Vitamin A Analysis: 
As an additional verification of the method, a series of frozen serum 
samples were analyzed with both conventional HPLC and HPCZE. The results 
from both techniques are listed in Table I. 
TABLE I 
______________________________________ 
Comparison of concentrations of retinol (Vitamin A) in human 
serum samples determined by HPLC and HPCZE 
Serum Concentrations of retinol (ng/mL) 
samples HPLC method.sup.1 
CZE method.sup.2 
______________________________________ 
1 463 445 .+-. 28 
2 177 185 .+-. 24 
3 231 268 .+-. 29 
4 404 424 .+-. 27 
5 216 206 .+-. 23 
6 498 428 .+-. 24 
7 204 270 .+-. 27 
8 386 376 .+-. 25 
______________________________________ 
.sup.1 The data came from Department of Nutritional Science, University o 
Connecticut. 
.sup.2 The data were the mean of 5 analysis .+-. standard deviation of th 
mean. 
Linear regression (forced through the origin) of the correspondence between 
the two methods gave slope 0.981 with standard deviation 0,041 (i.e., not 
statistically different from 1) with correlation coefficient 0.925. The 
average coefficient of variation (standard deviation divided by mean) for 
CZE analysis was 1.5%. However, the CZE method is much faster and easier 
than the HPLC method. 
EXAMPLE 2 
HPCZE AND DRIED BLOOD SPOT SAMPLES 
Serum and Blood Spot Sample Preparation: 
All fasting blood samples were obtained from volunteers in Northeast 
Missouri State University and Kirksville College of Osteopathic Medical 
Center. After clotting at room temperature in the dark, sera were prepared 
by Centrifugation. The blood spots were prepared by spotting the vein 
blood or finger-prick blood on 903 special filter papers (Schleicher & 
Schuell, Keene, N.H., USA) immediately after the blood was drawn. The 
blood was allowed to dry and then analyzed for retinol levels. 
Pretreatment of Blood Spot Sample: 
The blood spot on the filter paper was cut off from the center with a 1/4 
inch diameter paper hole puncher, and was put into a 1.5 mL 
microcentrifuge tube and 300 .mu.L ice-cold sample pretreatment buffer was 
added. Then the mixture was mixed on a Vortex mixer for 3-5 min 
*uncontinuously) until the blood spot was dissolved in the buffer 
completely. Then 100 .mu.L of the blood spot solution was taken and 
filtered in the same way as that of serum sample. The filtrates were ready 
for HPCE analysis. 
Pretreatment of Serum Samples: 
To 10 .mu.L serum, 290 .mu.L ice-cold sample pretreatment buffer was added, 
and the mixture was mixed well on a Vortex mixer (Fisher, St. Louis, Mo, 
USA). The solution was allowed to stand on ice for 3-5 min, and 100 .mu.L 
solution was taken out and put into a Microcon-30 filter unit (Amicon, 
Inc., Beverly, Mass., USA) (this filter unit allows molecular weight &lt;30 
KDa molecules to pass through), and filtrated by centrifugation. The 
filtrate was ready to be injected into HPCE column for analysis. 
HPCZE Analysis: 
Pretreated serum samples were injected electrokinetically at 10 kV for 10 s 
(approximately 8-10 nl was injected), and the separation was carried out 
at 20 kV for 5-6 minutes. The electrophoretic current was monitored with a 
multimeter throughout the separation to ensure the reproducibility. After 
each run, the capillary was washed with running buffer. 
Conventional Extraction and HPLC Analysis of Retinol From Serum: 
To 100 .mu.L freshly thawed serum was added an equal volume of methanol 
containing an internal standard (retinyl acetate); each sample was 
extracted three times with an equal volume of hexane. Hexane from combined 
hexane extracts was evaporated under a gentle stream of Argon, and the 
residue was dissolved in 25 .mu.L 2-propanol:dichloromethane (4:1 by 
volume; 20 .mu.L was injected for HPLC analysis. Samples were analyzed by 
reversed-phase HPLC on a Resolve 5- .mu.m c.sub.18 column (Waters 
Associates, Milford, Mass.) using a mobile phase of methanol:water (95:5) 
at a flow-rate of 1.0 ml/min, with detection at 325 nm (25). 
Quantification of Retinol in Dried Blood Spot: 
There are two ways to quantify the retinol in the dried blood spot. In the 
first method, a calibration curve was set up with a series of standard 
blood spots whose serum Vitamin A concentration were known (analyzed by 
conventional HPLC method). Then the concentration of unknown blood spots 
can be found from the calibration curve. The second, one single blood 
spot, whose retinol concentration was quantified by HPLC, was used to make 
a calibration curve by diluting to different concentration of retinol 
after filtration. The Vitamin A concentration of unknown blood spots also 
can be found out from the calibration curve. 
FIG. 4 shows the electrophoregram for direct injection of the dissolved 
blood spot sample without filtration under the current electrophoretic 
conditions. It is clear that it is impossible to determine retinol 
quantitatively. This is mainly caused by capillary wall adsorption of 
proteins and other molecules from the blood (the blood cells were broken 
during the dissolving process). Therefore, we adopted a membrane filter 
unit to get rid of most of the large size molecules from the blood sample. 
This step greatly reduced the wall adsorption problem and increased the 
reproducibility. The capillary was washed with running buffer between 
runs. The function of 6M urea is to totally dissociate plasma 
transthyretin (TTR) from retinol-RBP. This sample pretreatment step can 
also be applied to serum samples. The electrophoregram in FIG. 2 shows the 
separation of retinol-RBP in the pretreated serum sample. It can be seen 
that the matrix affects have been greatly decreased and the peak can be 
accurately integrated by integrator. We need to point out that the first 
peak in FIG. 5 was not coming from serum sample. We have done a blank 
control experiment, which allowed only pretreatment buffer to be filtrated 
with the filter unit by the same way with that of the blood sample, we 
still got that first peak, shown in FIG. 6. It was proven that the first 
fluorescence signal came from the filter unit. 
FIG. 7 shows the calibration curve from 10 serum samples. The concentration 
of retinol has been determined with HPLC. The calibration curve was made 
with the sera whose vitamin A concentrations are known. When these sera 
are injected into the HPCE column, the higher concentration of vitamin A 
will give a higher signal (i.e., will give a larger peak area), the lower 
concentration of vitamin A will give a smaller signal. Based on these peak 
areas according to different vitamin A concentration, a curve of 
responding signal (peak area) vs vitamin A concentration can be made. When 
an unknown serum sample is injected for analysis, a peak area of 
retinol-RBP for this serum sample can be obtained. From the calibration 
curve, we can find out the concentration of vitamin A corresponding to 
this peak area in this unknown serum sample. It can be seen that 9 samples 
show a linear response between fluorescence signal (peak area) and retinol 
concentration. However, one sample shows a much lower signal than other 
serum samples. The electrophoregram for this sample was also a little 
different from others (it was shown in FIG. 8), and a peak shows up right 
after the retinol-RBP peak. This could be due to human variability because 
some people may have different retinol metabolism and may have another 
retinol transport protein in serum. 
FIG. 9 shows the separation of retinol-RBP from dried blood spot. It can be 
seen that more peaks were observed from blood spot samples than the serum 
samples. This means that some molecules in the non-serum component of the 
blood spot may also fluoresce at the same wave length as that of 
retinol-RBP. In order to quantify the retinol in the dried blood spot, we 
used one blood spot, whose serum retinol concentration has been determined 
by using HPLC, to make a calibration curve by diluting to different 
concentration, the curve was shown in FIG. 10. The concentration of other 
blood spot samples can be found out from this calibration curve. Table II 
shows the retinol concentration of 10 blood spot samples found out from 
the calibration curve, and comparison to the value found out from HPLC. 
TABLE II 
______________________________________ 
Comparison of results between HPLC and HPCE for blood spot 
retinol analysis (Notes: the HPCE results were found from the 
HPCE calibration curve) 
Retinol Concentration 
Retinol Concentration 
Sample # by HPLC by HPCE 
______________________________________ 
1 73.8 73.7 
2 35.2 39.0 
3 39.2 39.8 
4 49.4 55.1 
5 62.0 33.8 
6 78.0 79.3 
7 49.6 53.0 
8 59.4 58.8 
9 59.6 65.0 
10 57.8 60.7 
______________________________________ 
It is clear that 9 blood spot samples match with the HPLC data well and one 
blood spot sample was off, this affect was also seen in the serum data. 
Since the 10 serum samples and 10 blood spot samples were coming from the 
same 10 people, we can see that the results from blood spots were very 
close to those of serum samples. 
In order to quantitatively determine the retinol in dried blood spot, we 
have done a recovery study from 903 filter paper. A known volume of serum 
was spotted on the filter paper. After being dried, the whole spot was cut 
off and treated the same way as those of blood spot and analyzed by HPCE. 
Same volume of serum was taken and analyzed by HPCE without spotting on 
the filter paper. The recovery was calculated based on the peak area. The 
recoveries of seven serum samples are listed in Table III, with a range of 
89% to 113%. 
TABLE III 
______________________________________ 
Recovery study of retinol in dried blood spot by 
high performance capillary electrophoresis 
blood spot sample 
percent recovery 
______________________________________ 
1 113.5 
2 107.6 
3 98.6 
4 90.1 
5 99.6 
6 96.8 
7 89.0 
______________________________________ 
From Table III we can see that for 7 samples studied, 6 of them have a 
percentage recovery over 96%. If an increase in the stirring time would 
help achieve even higher blood spot recovery in the pretreatment buffer. 
From the above examples, it can be seen that the invention accomplishes at 
least all of its stated objectives.