Abstract not afford the high cost of some


is the most prevalence inborn error in aminoacid metabolism. Its diagnosis
and monitoring depends on the quantification of phenylalanine in the blood.
Several analytical methods are used for that purpose. In this review, four
analytical techniques are presented, and a general comparison is discussed.
Several differences are noticed between them depending on their validation

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Phenylalanine    Phe   
Phenylketonuria    PKU    Tandem mass spectrometry    MS/MS     High      
performance liquid chromatography  
Enzyme chip Gas chromatography / Mass spectrometry    GC/MS



(PKU) is the most prevalent inborn error in aminoacid metabolism in the world (Blau, van Spronsen, & Levy,
2010). It is an autosomal recessive disorder
caused by the deficiency in activity of phenylalanine hydroxylase, which cause
impaired conversion of phenylalanine (phe) to tyrosine (De
Silva, Oldham, & May, 2010).

Phe is an amino acid with the formula C9H11NO.
This is classified as neutral, and nonpolar because of the hydrophobic nature of the benzyl side chain (Bhagavan & Ha, 2011).

Methods for PKU detection include: bacterial inhibition
assay, fluorimetry, enzymatic method, high-performance liquid chromatography (HPLC),
ion-exchange chromatography, gas chromatography or tandem mass spectrometry
(MS/MS) (Kand’ár & Žáková, 2009).

The Guthrie
method using a bacterial inhibition assay, is the universal diagnostic
procedure, but it requires long incubation time, and it is sometimes fails to
record accurate results (Tachibana, Suzuki, & Asano, 2006).

Untreated children with PKU are normal at birth,
but soon they don´t attain normal development (Koch et al.,1984). Early
diagnosis of PKU prevents mental disability, and appropriate control of blood
phe is critical for the patient to avoid most of the CNS diseases caused by PKU
(Blau et al., 2010).

Many countries could not afford the high cost of some methods for PKU diagnosis. It was possible to save a significant number of children with mental retardation by PKU early detection (Mihali, 2017).

In this review,
four techniques for phe measurement in blood will be presented and compared by
their advantages and limitations in order to determine the most suitable method
for phe measurement and PKU diagnosis.


High Performance Liquid Chromatography (HPLC)

The research that was done by HPLC for phe measurement mainly use dry
blood spot or blood serum as a specimen. The detection was performed using UV
light or Florescence.

In a previous study, plasma from
blood was collected and prepared by centrifugation. Amino acids
were separated by deproteinization of plasma, filtration of sample, followed by
ion-exchange chromatography. The eluate was evaporated in vacuum at 40°C, and the residue was
dissolved in distilled water. The final extract contained a concentrated
solution of amino acids free of interfering substances. HPLC analysis
was reversed phase, with isocratic elution, and the elution time was 7 minutes. Each sample was
injected four times, the results showed small sample to sample differences and
small standard deviation value (0.01). The linearity of the values of  the calibration curve with the standard
solutions from the lowest concentrations (109 ?mol/L) up to very high
concentrations (1834.4 ?mol/L) (Ladasiu, 2017). This method delivers a quick and a cheap alternative to those using
HPLC with  derivatization (Danafar & Hamidi, 2015). 

The advantages
of using dried blood spot (DBS) as specimen has been highlighted recently
because it is noninvasive and requires very small amount of sample (few
microliters). In a previous study, serum samples are collected and separated
from formed elements by centrifugation then ultrafilteration. A disk containing
3.3 ?L of blood was punched from each DBS. Then, cold methanol was added. The
samples, after being mixed by vortex mixer, were centrifugated. The supernatant
was transferred into conical vial inserts for precolumn derivatization and then
for HPLC analysis. Reversed phase HPLC, with gradient elution was used. The
total elution time was 20 min. To establish intra- and inter day coefficients
of variation, five replicates of DBS at four different concentrations of phe
were performed within the same day and in five consecutive days using a daily
prepared calibration curve. The assessed CVs were (below 7%) for all
concentrations. Recovery rates were between (93 and 101%). The LOD determined
for was (0.1 ?M), while the LOQ was (12 ?M) (Pecce, Scolamiero, Ingenito,
Parenti, & Ruoppolo, 2013).

In order to overcome some disadvantages, including
complex derivatizing agents and sample preparation, another method was
conducted using the natural fluorescent properties of phe. DBS are easier
obtained and transported than liquid specimens. Blood-spot samples were collected onto 903
Specimen Collection Paper and allowed to dry for at least 24 h before
measurement of phe concentrations. Spots were immediately used or stored at 4 ?C. Reversed phase HPLC was used. The mixture of ethanol and
deionized water was used as a mobile phase with isocratic elution. The mean spike recovery was (97.1%). The
calibration curve was linear in the whole range tested: (10.0- 1500.0 mol/L).
The correlation coefficient for the calibration curves was (0.9997). The limit
of detection was (10.0 mol/L) (Kand’ár
& Žáková, 2009).

In order to
confirm the suitability of HPLC technique for phe measurement, another study
was conducted using case and control subjects` blood samples. In a control
subject (PKU free), the concentration of plasma phe was (43 ?mol/L). This is a
very small value, and confirms that the HPLC method is very sensitive in
evaluating the plasma Phe concentration. In a PKU subject, the concentration
of phe was (2496 ?mol/L), which is higher than the highest concentration of the
standard solution (1600 ?mol/L). When the plasma extract from PKU patient was
diluted 1:1 with water, the value of plasma phe for the diluted sample appeared
to be (1242 ?mol/L), which is close to half of the concentration of the undiluted
sample (2496 ?mol/L). This confirms the high precision of the HPLC for phe measurement (Mihali, 2017).

HPLC method
presents several advantages: non expensive, simple, sensitive and accurate. Any
laboratory having a HPLC instrument can set up the procedure easily (Mihali, 2017). HPLC is an adequate technique for the diagnosis of PKU, and also
for monitoring the plasma concentrations of phe in patients with PKU (Ladasiu, 2017). Using DBS, the sample can be easily withdrawn even at home, with
stability. It is an ideal tool in areas where obtaining large blood sample is
not possible. The method can be used as a validated assay in routine follow-up
of PKU patients being a valid and fast test alternative to common serum HPLC
method (Pecce et al., 2013).


Tandem mass spectrometry (MS/MS)

Using two mass
spectrometers in tandem MS/MS enables control of the formation of molecular and
fragment ions. The first mass spectrometer measures the mass of intact
molecules, fragments them, and the second mass spectrometer measures the mass
of the fragments. Fragments in the second mass spectrometer can be correlated
with the intact molecules produced in the first one (Chace & Kalas, 2005).

In a previous
study, MS/MS was used using DBS specimen. Whole blood was spiked with
phenylalanine, then, bloodspots were prepared by pipetting a single drop of
blood on 903 paper. For MS-measurements, a Quattro Ultima triple quadrupole
mass spectrometer (tandem MS) interfaced with an electrospray ionization (ESI)
source and equipped with an Alliance 2795 HPLC was used. The MS was operated in
positive ESI mode.  Injection volume was
5 ?L.  Masslynx software was used for
instrument control, data acquisition and data processing. m/z= 140.17. Results from this experiment showed a good correlation even at high
concentrations of phenylalanine (r2 =0.9917). LOD was (2 ?mol/L) and LOQ was (4 ?mol). Within run variations was (8.1%) and between run variations was (12.0%) (Prinsen, Holwerda-Loof, de Sain-van
der Velden, Visser, & Verhoeven-Duif, 2013).

Another study
was conducted in order to make a validation of the PKU screening with MS/MS. It was performed using in-house prepared standards and commercial
controls. The within-run variation was examined by 20 repeat measurements of
samples with two phe concentrations. The between-run variation was determined
by measuring the same samples on 20 consecutive days. For evaluation of sensitivity, specificity, predictive value and accuracy,
data from the measurement of 10136 dried blood samples from healthy newborns
was used. The results: CV% (6.8).
Recovery% (111). Detection limit (6 ?mol/l). Linear
range (6-1800 ?mol/l). Sensitivity% (100). Specificity% (98.9). Diagnostic
accuracy% (98.9) (Ceglarek et al., 2002).

Some PKU patients or parents cannot
produce a bloodspot that meets the quality requirements for reliable analysis. In
this technique, the reduction in bloodspot volume, is patient friendly and is time
saving in sample preparation depending on the number of samples analyzed (Prinsen et al., 2013). MS/MS
does not require secondary injections, separate sample analysis, or
modifications in sample preparation (Chace
& Kalas, 2005).


Gas chromatography – Mass spectrometry (GC/ MS)

In GC/MS, the analytes are derivatized before analysis
in order to facilitate chromatographic separation (Halket, J et al., 2004). GC/MS
using blood spot specimen
for the quantitative analysis of phe requires propyl chloroformate derivatization or microwave-assisted

In a previous study, whole blood was collected from patients with
PKU using a filter paper technique. Amino acids were extracted from the sample,
the extracts were purified using cation-exchange resins. The isotope dilution
method using 2H5-Phe as internal standards was applied. Propyl chloroformate
derivatization was conducted and the derivatives were analyzed using GC/MS. GC injection
temp. was 280 ?C, and the carrier gas was He. MS selective
ion monitoring (m/z=190). Calibration curves at concentrations from (0.0 to 1666.7
mol/l) with regression coefficient (R2= 1.000). The LOD= (12.9 mol/l). Retention
time= (2.86 min). Repeatability %RSD= (14.1). Recovery= (79.0%). From these results,
total analysis time= (80 min), include 60 min extraction time, 10 min purification
and derivatization time and  10 min GC/MS
analysis time that include column cool-down (Kawana,
Nakagawa, Hasegawa, & Yamaguchi, 2010).

In another
study, a method based on microwave-assisted silylation followed by (GC/MS) was
conducted. The amino acids were derivatized with N,O
bis(trimethylsilyl)trifluoroacetamide (BSTFA) under microwave irradiation. The
results showed that microwave irradiation can accelerate the derivatization
process, and shorten analysis time. MS was in the electron impact (EI) mode. He
(99.999%) carrier gas was used in GC, in retention time = (11.38 min.). The
hole analysis time WAS (less than 40 min). R2= (0.992). Precision %= (8.06). Detection
limit mM= (0.48). Recovery %=(98) (Deng, Yin, Zhang, & Zhang,

has a high resolution, and the standardized electron impact ionization
conditions enables establishment of searchable mass spectra libraries (Xiong et al., 2015). The derivatization reaction usually
performed with conventional heating, which requires long time (more than 30
min). Therefore, in the GC/MS screening procedure, it is preferred to use a
fast derivatization technique such as
microwave irradiation derivatization (Deng et al., 2005).


Enzyme chip

The use of enzyme
chip is a new microquantification method applied for measurement of phe
concentration in a dried blood spot.

In a previous
study, enzyme chip immobilized with His-tag fused phenylalanine dehydrogenase
(PheDH) was developed. His-tag fused PheDH was immobilized on the surface of
nickel-coated slide glass. A microarray sheet was made with
poly(dimethyl siloxane) (PDMS) using the photolithographic technique. Two types of PDMS microarray sheets were prepared and placed on top
of each other, adhered by compression without glue, and used as the enzyme
reaction chamber sheet. A 3-mm (diameter) disk was punched from a dried blood
spot on filter paper and put into each well of a 96-well flat-bottomed black
microplate. To extract phe and other soluble components, an extraction buffer
was added to each well and the microplate was left for 1 h at room temperature
with the top of the plate sealed to prevent vaporization. Each extract was
transferred into a microtube, NAD+ and diaphorase from Clostridium kluyveri
dissolved in potassium phosphate buffer, pH 7.5, were added. The mixture was
poured into the enzyme reaction chamber constructed with PDMS sheet on an
Ni–NTA-modified slide glass and sealed with a cover glass.  The enzyme reaction was performed at 25 °C
for 1 h, followed by fluorometric scanning at 532nm of excitation wavelength
and 585 nm of emission wavelength using Hitachi Software. The fluorescent
intensity was analyzed with DNAsis array software (Hitachi Software).  Linear calibration curve was obtained for Phe
in the range of (0 to 12.8mg/dl) with the dried blood spot filter papers. CVs
ranged from (0.3 to 14.2%) in the concentration range from (0.4 to 12.8mg/dl)
in the dried blood spots. Precision of the assay was calculated by replicate
analysis of the same spot sample. The linearity of the chip was conformed up to
(12.8mg/dl) for phe and had good correlation, r^2= (0.992). This work
showed that the His-tag-fused PheDH-immobilizing enzyme chip is applicable for
the diagnosis of PKU (Tachibana et al., 2006).

In another study, amino acid quantification was
made by loading new amino acid metabolic enzymes onto chips to enable the
measurement of the concentration of each amino acid. The data of a quantified
amino acid was compared with data obtained from the other methods of amino acid
analysis and showed good results (Asano,

The use of enzyme
chip can be applied to the diagnosis of PKU because it showed the same accuracy
and reliability as with the current PKU diagnosis kit (Tachibana et al., 2006).


Results and Discussion  

The aim of this
paper is to make comparisons between four advanced analytical techniques for
phe measurement in blood. The techniques are: HPLC, MS/MS, GC/MS and enzyme
chip. The results were collected from various previous studies. Different
application strategies were conducted with different specimen types even within
the same technique. The validation data from the most developed application
method within every technique was collected. After the correction of the units
(?mol/l), the data is presented in the table below.





Linear range

Correlation coefficient




days  RSD


HPLC (DBS & florescence)

97.1 %

10- 1500 ?mol/l


10.0 ?mol/L

3.3 – 4 .1 %

6.9 – 8.5 %

50 min.


111 %

6 – 1800

6 ?mol/l




<40 min. GC/MS (microwave-assisted silylation) 98 % 20 – 800 ?mol/l 0.992 0.48 ?mol/l - 8.6 % - 40-50 min. Enzyme chip - 0 - 774.9  ?mol/l 0.992 24.21  ?mol/l - 0.3 - 14.2 % - >1 hour


From the table,
it is noticeable that there are many variations between methods` recoveries,
LODs, precisions, and total times. However, some data for some methods was not

HPLC has the
best correlation coefficient and the least RSD % range, this indicates that
HPLC method is the most accurate and precise one. However, it needs long time.

MS/MS has the
wider linear range, it detects phe in various blood levels, and it requires the
shorter total analysis time.

GC/MS has the
best recovery percent, and the smallest LOD, that indicates that it is the most
sensitive method.

Using enzyme chip for phe measurement is not sensitive.
But, it has a wide RSD%, it could be the most or the least precise method.
Moreover, it requires the longest time.

Due to its
short time, MS/MS become the screening tool for PKU diagnosis, but it is
expensive and requires a special technicians and it is not suitable for the routine monitoring of
Phe in PKU patients (Deng et al., 2005).

On the other hand, HPLC with florescence is used for the follow up of a diet therapy in PKU patients (Kand’ár & Žáková, 2009).

In addition,
GC/MS is a simple, inexpensive, and is widely used for blood phe quantification
 (Deng et al., 2005).

methods, such as a bacterial inhibition assay, fluorometric detection, and
enzymatic colorimetry assay, have been applied in diagnosis of PKU due to their
simplicity and low cost. However, these methods also have some disadvantages of
low accuracy and low sensitivity (Xiong
et al., 2015). Conventional semi quantitative microbioassay had been
also used, but it required very long time -6 hours- (Matsunaga et al., 1981).

PKU patients have to monitor their blood phe over their
entire lifespan. It is known that PKU patients would benefit significantly if
they were able to monitor their Phe concentrations simply an economically at
home on a regular basis (Allard et al., 2004).

More research
are needed to compare the measurement of phe concentrations using different
techniques; a better understanding of differences in analytical methods is a
key to develop guidelines for clinicians and metabolic nutritionists who work
with PKU patients (Gregory, Yu, & Singh, 2007).


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