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J. Sep. Sci. 2012, 35, 34273433

Ana Valente1

Maria Rosario
Bronze2,3
Manuel Bicho4,5
Rui Duarte6
Helena Soares Costa1
1 Department

of Food and
Nutrition, National Institute of
Health Dr. Ricardo Jorge,
Lisbon, Portugal
2 Faculty of Pharmacy, University
of Lisbon, Lisbon, Portugal
3 Instituto de Tecnologia Qumica

e Biologica,
Oeiras, Portugal
4 Genetics Laboratory,Faculty of
Medicine, University of Lisbon,
Lisbon, Portugal
5 Instituto Rocha Cabral, Lisboa,
Portugal
6 Portuguese Diabetes
Association, Lisbon, Portugal
Received July 13, 2012
Revised August 24, 2012
Accepted September 4, 2012

Research Article

Validation and clinical application of an

UHPLC method for simultaneous analysis
of total homocysteine and cysteine in human
plasma
Several studies indicate that high levels of homocysteine (Hcy) and L-cysteine (L-Cys) are
independent risk factors for cardiovascular disease. The validation and clinical application
of an ultra HPLC method for analysis of Hcy and L-Cys is described. The reported method is
simple, sensitive, rapid, precise, and less aggressive than other previously reported methods.
The effect of the derivatization reaction time, pH, and organic solvent contents in the mobile
phase are described and discussed. Optimized conditions resulted in excellent peak shapes.
Results of method validation showed a good linearity (r2 0.993) over the investigated
concentration ranges and were observed for both compounds. The LOD and LOQ were
0.05 M and 0.15 M for Hcy and 0.24 M and 0.80 M for L-Cys, respectively. Validation
results proved that the method precision was good and the accuracy was satisfactory. This
validated method was successfully applied in an epidemiological study to measure and
compare the prevalence of Hcy and L-Cys high levels in plasma of Portuguese type 2
diabetic patients with and without angiopathy. The study results showed that prevalence
of hyperhomocysteinemia and hypercysteinemia were at least two times higher in diabetic
patients with angiopathy compared to diabetics without angiopathy.
Keywords: Cysteine / Homocysteine / Ultra HPLC / Validation
DOI 10.1002/jssc.201200672

1 Introduction
Homocysteine (Hcy) and L-cysteine (L-Cys) are nonessential,
sulphur-containing amino acids formed during the methionine metabolism. Hcy is present in several forms in the human
plasma. The concentration of total Hcy in human plasma is
defined as the sum of all Hcy species, including free, proteinbound, and oxidized forms, although it is not known which
form is directly involved in pathological processes [14].
Results from several case-control and prospective studies
indicate that moderate hyperhomocysteinemia and hypercysteinemia are independent risk factors of cardiovascular disease [511]. In a clinical setting, measurements of plasma Hcy
and L-Cys are made for a variety of purposes, such as cardiovascular disease risk assessment. Few studies have investiCorrespondence: Dr. Helena Soares Costa, Department of Food
and Nutrition, National Institute of Health Dr. Ricardo Jorge, I.P.,
Av. Padre Cruz 1649016 Lisbon, Portugal
E-mail: helena.costa@insa.min-saude.pt
Fax: +351-217508153

Abbreviations:
ABD-F,
4-aminosulfonyl-7-fluoro-2,1,3benzoxadiazole; ANOVA, analysis of variance; Hcy,
homocysteine; L-Cys, L-cysteine; SBD-F, 7-fluorobenzo2-oxa-1,3-diazole-4-sulphonate;
TCEP,
tris-(2-carboxylethyl)-phosphine; UHPLC, ultra high-performance liquid
chromatography


C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

gated the independent association between high circulating

levels of L-Cys and cardiovascular disease, but according to
these studies [12, 13] hypercysteinemia is a risk factor for cardiovascular disease. Thus, determination of total plasma Hcy
and L-Cys levels is essential to understand the role of these
compounds in the pathogenesis of cardiovascular diseases.
Several studies have documented plasma Hcy and L-Cys
concentration in healthy and patient subjects [1416], but
to our knowledge, there is no literature presently available
on total plasma Hcy and L-Cys levels in Portuguese type 2
diabetic subjects.
The clinical interest of Hcy and L-Cys as cardiovascular
markers has justified the need to develop rapid, precise, and
accurate techniques [1720]. Several types of analytical methods can be applied in plasma Hcy analysis, such as GC-MS
or LC-MS, CE, IA, ion change chromatography, and HPLC.
For clinical practice, the choice of an HPLC method is more
suitable than the other described methods, because it gathers several advantages. This technique has a lower cost when
compared with LC-MS or GC-MS, is more sensitive than
CE method, and allows detection of other aminothiols in
the same analysis, which is not possible with the IA method.
The use of HPLC as a separation technique has often been
the method of choice for the detection of total Hcy, as it can
be readily automated and easily coupled with various postcolumn detection methodologies [21]. The HPLC method with
fluorescence detection for Hcy and L-Cys analysis in plasma

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J. Sep. Sci. 2012, 35, 34273433

A. Valente et al.

is perhaps the most used end-column detection methodology due to its high sensitivity and reliability compared to
UV-Vis and electrochemical detections [21]. In HPLC techniques, the choice of a reducing agent has been a source
of controversy during the last years. The most commonly
used reduction agent is tri-n-butylphosphine [22, 23], but it
is a harmful substance with an unpleasant odor, and it is
poorly soluble in water. Therefore, it has been recently suggested that tri-n-butylphosphine should be replaced by tris(2-carboxyl-ethyl)-phosphine (TCEP), which is a nonvolatile,
stable compound, soluble in aqueous solutions and it is not
necessary to prepare it under nitrogen atmosphere under
controlled conditions [24]. The most frequently used derivatizing agents are the halogen sulfonyl benzofurans: ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-F),
and 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (ABD-F)
because of their good Hcy adduct stability and high HPLC resolution. These derivatizing agents are not fluorescent, their
thiol adducts are stable, and fluorescent hydrolysis products
are not formed. Thus, their use allows isocratic separation,
resulting in very clean chromatograms with no interfering
peaks. Although, SBD-F is less-reactive than ABD-F, detection of the fluorescent SBD-F derivative after HPLC is currently used in HPLC assays for plasma Hcy analysis, at least in
part because SBD-F adducts show a markedly lower retention
time than ABD-F adducts.
In this paper, the development and validation of an ultra
high-performance liquid chromatography (UHPLC) method
for simultaneous analysis of Hcy and L-Cys in human plasma
is described. This simple, sensitive, rapid, and less aggressive assay is an excellent choice for epidemiological studies
and for clinical analysis routine. The present validated UHPLC method has been applied in an epidemiological study to
measure and compare the prevalence of high Hcy and L-Cys
plasma levels in type 2 diabetic Portuguese patients with and
without angiopathy.

2 Materials and methods

2.1 Chemicals and solutions
DL-Hcy, EDTA, and potassium dihydrogen phosphate were
obtained from Sigma (St. Louis, MO, USA). TCEP and SBDF were acquired from Sigma or Fluka (Buchs, Switzeland);
L-Cys was supplied by Panreac (Montcada, Barcelona, Spain),
while ACN HPLC grade was bought from Carlo Erba Reagenti
(Rodano, Italy). All other reagents were obtained from Merck
(Darmstadt, Germany). All solutions were prepared with water from a Milli-Q ultrapure water purification system (Millipore, Bedford, MA, USA).
TCEP (174 mM) and the derivatization solution SBD-F
(4.25 mM) were prepared in ultrapure water. These two solutions were protected from light, divided into 40 and 60 L
aliquots, respectively, and stored at 80C. Trichloroacetic
acid solution (612 mM) was prepared by weighing 1 g of the
acid, 5.8 mg of EDTA, and dissolved in 10 mL of ultrapure

C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

water. The stock solution was sonicated for 15 min to ensure

complete dissolution. This solution was freshly prepared every week and kept at room temperature.
Sodium borohydride buffer solution (125 mM) with
EDTA (4.0 mM) was prepared by adding 2.52 g of sodium
borohydride and 117 mg of EDTA in 100 mL of ultrapure water and pH value was adjusted to 9.5 with sodium hydroxide
(1.5 M). This solution was freshly prepared every week and
kept at 4C.
2.2 Calibrators
Stock solutions of Hcy (600 M) and L-Cys (600 M) were
prepared by dissolving 4.1 mg of Hcy and 3.6 mg of L-Cys in
50 mL with a 5 mM HCl solution. Since L-Cys has a moderate solubility in aqueous HCl solution, it was necessary to
sonicate the stock solution for 15 min to ensure complete
dissolution. Hcy and L-Cys stock solutions were separated in
aliquots and stored at 80C until the day of analysis.
The working standard solutions were prepared daily from
the stock solution by appropriate dilution with 5 mM HCl to
obtain final concentrations of Hcy (1, 10, 20, 30, 40, 50, and
60 M) and L-Cys (150, 175, 200, 225, 250, 275, and 300 M).
2.3 Plasma samples
Plasma samples were obtained from whole blood, collected
into vacuum EDTA tubes (Vacutainer tubes, Sarstedt, Portugal). Blood samples were immediately cooled, separated by
centrifugation at 2000 g for 15 min within 30 min after
venipuncture, and stored at 80C in microtubes (0.5 mL)
until analysis.
2.4 Analytical procedure
A total of 100 L of plasma or Hcy and Cys working standards were mixed with 30 L of 174 mM TCEP and incubated
for 10 min at 37C to reduce and release protein-bound thiols. Deproteinization was achieved by addition of 150 L of
trichloroacetic acid (100 g/L) solution followed by centrifugation (10 400 g for 10 min at 20C). The clear supernatant
(50 L) was mixed with 10 L of 1.55 M sodium hydroxide
solution, 125 L of 125 mM borate buffer at pH 9.5, containing 4 mM and 50 L of a 1 g/L thiol-derivatizing reagent,
SBD-F. Complete derivatization was performed at 60C for
40 min. After cooling at 4C for 10 min, the reaction mixture was filtered through a 0.22 m PVDF Millipore filter
R
UPLC
(Millipore) and 10 L were injected onto the Acquity
system.

2.5 Instrumentation and chromatographic conditions

Separation and quantification were performed with an
R
Acquity
UPLC system (Waters, Milford, MA, USA)
equipped with a SupelcosilTM LC-18-DB analytical column
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J. Sep. Sci. 2012, 35, 34273433

(150 mm 4.6 mm id, 3 m particle size) from Supelco

(Bellefonte, PA, USA) and a SupelcosilTM LC-18-DB guard
column (20 mm 2.1 mm id, 5 m particle size), Supelco
(Bellefonte). Fluorescence intensity was measured with excitation at 385 nm and emission at 515 nm, using an Acquity UPLC FLR detector (Waters). The detection signal was
recorded and the peak areas were quantified and processed
TM
with an Empower software version 2.0 (Waters).
The mobile phase consisted of 30 mM potassium dihydrogen phosphate, pH 2.1 (adjusted with orthophosphoric
acid 85%), and containing 40 ml/L of ACN. The mobile phase
was filtered through a 0.22 m GHP membrane, Pall filter,
(Gelman Laboratory, Canada) and then degassed for 30 min.
The total run time of analysis was 10 min at a flow rate of
1 mL/min. Column temperature was kept at 20C and autosampler at 6C.

2.6 Method optimization

The effect of the derivatization reaction time, in the incubator
at 60C, was studied. Three different reaction times (10, 40,
and 60 min) were tested. Results from three plasma samples
analyzed in triplicate were compared.
In order to optimize the mobile-phase composition, different percentages of ACN (48%) and phosphate buffer concentrations (30100 mM) were tested. The pH of the aqueous
phase was established at 2.1 because maximal fluorescence
intensity of Cys and Hcy adducts occur in an acidic medium
(pH 2.0).
R
UPLC sysTwo different HPLC equipments (Acquity

R
tem and Alliance HPLC system 2695Waters) were compared. Several parameters were tested: (i) injection volume
(10, 20, and 30 L); (ii) flow rate (0.8, 1.0, and 1.5 mL/min);
(iii) autosampler temperature (6, 8, and 15C); (iv) column
temperature (20, 25, and 30C).

2.7 Method validation

Previous to the validation procedure, the optimized method
was tested for system suitability, by calculation of the following parameters: plate count (N), tailing factor (T), resolution
(Rs), retention time (tr ), capacity factor (K ), and system repeatability. These parameters were calculated from replicate
injections of standards, then evaluated and compared with
the recommendation limits [2527].
The analytical method was validated according to Food
and Drug Administration or International Conference on
Harmonisation guidelines [2628] and the following parameters were determined: sensitivity, specificity, LOD, LOQ,
range, linearity, precision, and accuracy.
Analytical specificity was assessed by the absence of any
interference at the retention time of the peaks with interest
and the resolution factor between Hcy and L-Cys.
The LOD was considered to be the lowest analyte concentration, which gives an S/N ratio of at least 3:1. The achieved

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Liquid Chromatography

3429

value was confirmed by analyses of five independent standard

solutions at LOD concentration.
The LOQ was determined as the lowest concentration that
gives an S/N ratio of at least 10:1. The calculated LOQ was
validated by analyzing, in triplicate, five independent standard
solutions at LOQ concentration.
Calibration curves were performed daily using seven standard concentrations, injecting six times each, for both analytes. Linearity of calibrations curves was evaluated by linear
regression analysis, plotting peak areas versus standard concentrations. Parameters like regression, determination coefficient (r2 ), slope, and intercept were calculated. The slope of
the calibration curve was also used to evaluate the method
sensitivity.
The intra-assay precision was investigated by injecting
in duplicate, six samples from a plasma pool with unknown
concentration, on the same day. The inter-assay precision was
evaluated on three different days by preparing six samples
from the same plasma pool on each day. Mean, SD and CV
were calculated, for both analytes.
The accuracy was calculated by analyzing a plasma pool
before and after addition of a known concentration of Hcy
and L-Cys. The recoveries were measured after spiking
samples with three independent standard solutions of Hcy
(1, 30, and 60 M) or L-Cys (150, 225 and 300 M) on three
different days. The measurements were performed by using
five determinations per concentration. After addition of standard solutions, the samples were processed according to the
procedure described above (Section 2.4).

2.8 Clinical application

The validated UHPLC method was successfully applied in
an epidemiological study to measure and compare the prevalence of Hcy and L-Cys high levels in plasma of Portuguese
type 2 diabetic patients and nondiabetic subjects. A total number of 150 type 2 diabetic patients were recruited from the
Portuguese Diabetes Association and 150 healthy adults were
recruited from the International University for Seniors. Subjects were invited to participate by phone or personal invitation. From all the 300 participants, only seven subjects gave
up during the study. The population was categorized in three
groups: group I (n = 75) was formed by type 2 diabetic patients
with angiopathy, group II (n = 75) by type 2 diabetic patients
without angiopathy, and group III (n = 143) by nondiabetic
subjects. The following inclusion criteria were applied to all
the groups: (i) written informed consent; (ii) age 4075; and
(iii) Caucasian. Inclusion criterion for groups I and II was
type 2 diabetes diagnosis for at least 1 year. Inclusion criterion for Group I was the presence of at least one of the following angiopathic complications: macroangiopathy (heart disease, stroke, peripheral vascular disease) or microangiopathy
(retinopathy). For group II, diabetic patients without cardiovascular complications described above were selected. The
presence of all angiopathic complications was confirmed by
the access to the patient medical records. Exclusion criteria for
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J. Sep. Sci. 2012, 35, 34273433

A. Valente et al.

group III are the presence of diabetes, metabolic syndrome,

or cardiovascular disease. The study was performed according to the Declaration of Helsinki (1975) and was previously
approved by three Ethics Committees: National Institute of
Health Dr. Ricardo Jorge, Faculty of Medicine from Lisbon
University, and Portuguese Diabetes Association.

2.9 Statistical analysis

Statistical analyses were performed with SPSS for Windows,
IBM SPSS Statistics 20.0 (SPSS Inc., Chicago, IL, USA).
Results are expressed as mean SD or as percentage.
Comparison between groups of numeric variables normally
distributed was performed by one-way analysis of variance
(ANOVA). Scheffes post hoc tests for multiple comparisons
were used to determine which mean is significantly different from the others. Bivariate associations between numeric
variables were assessed using Pearson correlation coefficient.
Statistical significance was considered for a p < 0.05. Hyperhomocysteinemia was defined for Hcy levels 15 M and
hypercysteinemia was defined for L-Cys > 300 M. Prevalence of high Hcy and L-Cys plasma levels was evaluated for
each group.

3 Results and discussion

der the mentioned conditions, the retention time for Hcy

and L-Cys was reasonable, while the capacity and resolution
factors were maximal. For all the tested phosphate buffer concentrations, no significant differences were observed; therefore a phosphate buffer concentration of 30 mM was chosen
because it was sufficient to maintain the pH value, extend
the equipment and column life times, and to encourage the
use of a less aggressive chromatographic method for routine
analysis. In the sample preparation procedure, the plasma
dilution is minimal and a low injection volume is used.
The optimal conditions, as determined from the results of
the experimental design were: (i) injection volume, 10 L;
(ii) flow rate, 1 mL/min; (iii) autosampler temperature, 6C;
and (iv) column temperature, 20C. The equipment with the
R
UPLC system. For the
best performance was the Acquity
same standard concentration and chromatographic conditions, the UPLC system equipment proved to be at least three
times more sensitive than the HPLC System 2695. Typical
chromatograms showing the elution profile of Hcy and L-Cys
in pure standard mixture and in human plasma are shown
in Fig. 1. Chromatographic runs of samples or standard solutions were performed isocratically within 10 min, with retention times of 4.0 min for L-Cys and 8.4 min for Hcy.
No interferences were observed in blank chromatograms simultaneously treated as plasma samples. The analytes were
identified on the basis of their retention times compared to
standard solutions.

3.1 Method optimization

3.2 Method validation
This paper describes the development of an UHPLC method
for measuring total Hcy and L-Cys in human plasma, using SBD-F and TCEP as a fluorescence marker and reducing
agent, respectively. The reduction agent chosen was TCEP
because it is nonvolatile, stable, and soluble in aqueous solutions and it also enables a rapid reduction (approximately
6 min). Thiol derivatization with SBD-F seems to be the best
choice because it is not fluorescent, no hydrolysis products are
formed, and fluorescent thiol products are stable. According
to the obtained results for the derivatization reaction times of
40 and 60 min, no significant differences in the peak areas
of Hcy and L-Cys were observed at a temperature of 60C
with SBD-F reagent. For the derivatization reaction time of
10 min, our results are not in agreement with the literature [29], since we observed lower peak areas of Hcy and
L-Cys, showing that reaction with SBD-F was incomplete.
Therefore, after method optimization, all analyses were performed with a reaction time of 40 min [22, 30]. The reaction
rate of thiols with SBD-F gradually increased with rising of
pH value [31]. High fluorescence intensities are observed for
Hcy in the range of pH 2 to 12, but for SBD-cysteine and SBDthiol-containing aminoacids, an acidic medium (pH 2.02.15)
is required [32]. According to this, the optimal pH for analysis
of the two thiol compounds was 2.1.
Different compositions of mobile phase were tested
to achieve the best elution of Hcy and L-Cys. The
ACN/phosphate buffer ratio was established at 4:96 v/v. Un
C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Chromatographic separation characteristics of Hcy and L-Cys

were determined in a standard chromatogram. Experimental
results were compared with the acceptance criteria (Table 1)
and all parameters were in agreement with the established
requirements [25].
In human plasma chromatograms, another peak close to
the Hcy peak was observed, but resolution factors in all chromatograms were always greater than the acceptance criteria
(Rs > 2.0).
The LOD was 0.05 M for Hcy and 0.24 M for L-Cys.
The LOQ was 0.15 M for Hcy and 0.80 M for L-Cys. The
limits were validated by injection of five standard solutions
at the detection and quantification limits concentration. Precision of quantification limit for both compounds were studied, although the method working range was higher than the
established limits. According to the guidelines recommendations [25], the calculated CVs were always lower than 20%.
The linearity of the method was proved for Hcy
and L-Cys over the used concentration range (Table 2).
The mean ( SD) regression equation from five
calibration curves on different days for Hcy was:
= 8600.11 (557.43)X + 2580.58 (1083.51), and for LCys: = 1773.25 ( 239.42)X + 8431.00 ( 3753.68). Determination coefficients (r2 ) were always equal or greater than
0.993, for both aminothiols. Slope variation coefficient (n = 5)
was lower than 15%. The ANOVA one-way test was performed
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Figure 1. Typical chromatograms of: (A) standard mixture with (1) 175 mol/L L-Cys and
(2) 10 mol/L Hcy; (B) plasma sample from a
type 2 diabetic patient containing (1) 342.7
mol/L L-Cys and (2) 23.7 mol/L Hcy, respectively; (C) plasma sample from a healthy subject containing (1) 165.3 mol/L L-Cys and (2)
7.2 mol/L Hcy, respectively.


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J. Sep. Sci. 2012, 35, 34273433

A. Valente et al.

Table 3. Intra-assay and inter-assay precision data

Table 1. System suitability data

Analytes

Parameters

Results

Acceptance
criteria

Hcy

Capacity factor
Resolution
Retention time
System repeatability
Tailing factor
Plate count
Capacity factor
Resolution
Retention time
System repeatability
Tailing factor
Plate count

K = 7.0
Rs = 9.5
tr = 0.83%
CV = 0.2%
T = 1.3
N = 3243
K = 2.0
Rs = 9.5
tr = 0.65%
CV = 0.9%
T = 0.9
N = 2459

K > 2
Rs > 2
tr 1%
CV 1%
T2
N > 2000
K 2
Rs > 2
tr 1%
CV 1%
T2
N > 2000

L-Cys

to prove variance homogeneity in the working range. Results indicate that for Hcy and L-Cys, no statistic differences
(p = 0.05) were achieved between all calibration curves. According to the LOD and LOQ and also to the linearity results,
this method was considered to be sensitive. The obtained results for LOD and LOQ are at least five times lower than the
reported limits in the literature [2, 30, 31, 33, 34]. According
to that, this method is suitable for Hcy and L-Cys analyses
in fetal blood samples. Intra-assay and inter-assay results for
both analytes are presented in Table 3. The method showed
to be precise for analysis of Hcy and L-Cys in plasma samples. The intra-assay CV (n = 6) for Hcy ranged from 2.14%
(8.38 0.18 M ) to 4.06% (8.81 0.36 M) and for L-Cys
from 2.35% (264.59 6.22 M) to 2.66% (255.10 6.79 M).
The inter-assay CV (n = 18) was 4.27% (8.19 0.35 M) for
Hcy and 3.07% (254.67 7.84 M) for L-Cys (n = 18). The results of the intra-assay and inter-assay precision show that CV
was always lower than the acceptance criteria established in
validation guidelines [26, 28] and proved to be in accordance
with previous data [24, 29, 30, 35].
The results of analytical plasma recoveries spiked with
10, 30, and 60 M of Hcy and 175, 250, and 300 M of
L-Cys are shown in Table 4. The method recoveries for Hcy
range from 91.6% (27.6 2.2 M) to 95.1% (57.1 1.4 M)
and for L-Cys from 90.0% (231.0 11 M) to 92.4%
(230.9 0.77 M). The recoveries obtained in standard addition experiments for Hcy and L-Cys were in agreement with
the previous findings [29, 30, 33, 35].

Analyte

Precision

Hcy

Intra-assay

L-Cys

Inter-assay
Intra-assay

Inter-assay

Day 1
Day 2
Day 3
Inter-day
Day 1
Day 2
Day 3
Inter-day

Mean SD (M)

CV(%)

8.02 0.18
8.81 0.36
8.38 0.18
8.19 0.35
255.10 6.79
264.59 6.22
241.44 5.94
254.67 7.84

2.22
4.06
2.14
4.27
2.66
2.35
2.46
3.07

Table 4. Validation data for method accuracy

Analyte

Spiked
(M)

Measured mean
SD (M)

Recovery
(%)

Hcy

10
30
60
175
250
300

9.67 0.84
27.6 2.2
57.1 1.4
162.1 1.6
230.9 0.77
231.0 11

93.6
91.6
95.1
91.4
92.4
90.0

L-Cys

3.3 Clinical application

The mean Hcy (10.61 4.80 M) and L-Cys (246.53
55.58 M) plasma levels for group I were higher than in
groups II (Hcy: 9.40 4.65 M; Cys: 229.43 48.08 M)
and III (Hcy: 4.10 2.16 M; Cys: 93.57 21.77 M). Oneway ANOVA (F 98.5, p = 0.00) application to test statistical differences in Hcy levels showed significant differences
among groups. Results of Scheffes test (p = 0.00) showed
that Hcy levels were statistical different between diabetic patients and nondiabetic patients. One-way ANOVA (F 480, p =
0.00) application to test statistical differences in L-Cys levels
showed significant differences between groups. Scheffes test
results proved that L-Cys levels were significantly different
between the three study groups. The prevalence of hyperhomocysteinemia was 20% for group I and 8% for group II.
The prevalence of hypercysteinemia was 17% for group I and
8% for group II. According to these results, the prevalence
of hyperhomocysteinemia and hypercysteinemia was at least
two times higher in group I than in group II.

Table 2. Linearity of UHPLC method for total Hcy and L-Cys analysis in human plasma

Analytes

Concentration
range
(M)

Slope
(n = 5)
Mean SD (M) (CV%)

Intercept
(n = 5)
Mean SD (M) (CV%)

Determination
coefficient
(r2 )
Mean SD (M) (CV%)

Hcy
L-Cys

160
150300

8600 557.4 (6.48)

1773 239.4 (13.50)

2.581 1084 (41.99)

8.431 3764 (44.64)

0.998 0.002 (0.15)

0.993 0.002 (0.19)


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3433

4 Concluding remarks

[12] Jacob, N., Bruckert, E., Giral, P., Foglietti, M. J., Turpin,
G., Atherosclerosis 1999, 146, 5359.

In summary, the current validated UHPLC method for simultaneous analysis of Hcy and L-Cys in human plasma is
rapid, specific, sensitive (LOD = 0.05 M for Hcy and LOD =
0.24 M for L-Cys), precise (CV <4.3% for Hcy and CV <3.1
for L-Cys) and accurate (recoveries: >92% for Hcy and >90%
for L-Cys). Furthermore, the method requires small sample
volumes (10 L) and has a short run time (10 min) without a need of a cleaning step, which is very useful for high
turnover during routine clinical analysis. Measurement of
both plasma Hcy and L-Cys has many clinical implications in
the diagnosis of cardiovascular disease, especially in type 2 diabetic patients. The validated UHPLC method has been used
to support an epidemiological investigation on hyperhomocysteinemia and hypercysteinemia prevalence. The presence
of angiopathy in Portuguese type 2 diabetic patients seems to
be related with the prevalence of hyperhomocysteinemia and
hypercysteinemia.

[13] El-Khairy, L., Ueland, P. M., Refsum, H., Graham, I. M.,

Vollset, S. E., Circulation 2001, 103, 25442549.

This study was financially supported by the research project

number 106/2007 from Comissao de Fomento da Investigacao em
Ministerio da Saude,
by the research project
Cuidados de Saude,
PIC/IC/82957/2007 from FCT and by Instituto Nacional de
Doutor Ricardo Jorge, I.P. (Lisboa, Portugal). Ana Valente
Saude
acknowledges the financial support received from FCT, through
the PhD grant (SFRH/BD/16166/2004/5E4M).
The authors have declared no conflict of interest.

5 References
[1] Still, R. A., McDowell, I. F. W., J. Clin. Pathol. 1998, 51,
183188.
S., Bilgi, C., Cakir, E., Erbil, M. K., Kutluay, T.,
[2] Tokgoz,
Turk. J. Med. Sci. 2003, 33, 7176.

[14] Lahoz-Rallo, B., Blanco-Gonzalez, M., Casas-Ciria, I.,

Marn-Andrade, J. A., Mendez-Segovia, J. C., MoratallaRodriguez, G., Quintero-Dominguez, R., Ramirez-Raya,
M., Guerrero-Pinedo, M. J., Aguilar-Diosdado, M., Diabetes Res. Clin. Pract. 2007, 76, 436444.

[15] Ndrepepa, G., Kastrati, A., Braun, S., Koch, W., Kolling,

K., Mehilli, J., Schomig,

A., Nutr. Metab. Cardiovasc. Dis.
2008, 18, 6673.
[16] Buysschaert, M., Preumont, V., Hermans, M. P., Diabetes
Metab. Res. Rev. 2007, 1, 5359.
[17] Ducros, V., Demuth, K., Sauvant, M. P., Quillard, M.,
E., Candito, M., Marie-Hel
ene,
`
Causse,
R., Drai, J., Garcia,
I., Gerhardt, M. F., J. Chromatogr. B 2002, 781, 207226.

Yaman, H., Bilgi, C.,

E. O., Cakir, E., Ozcan,
[18] Akgul,
O.,
Erbil, M. K., Turk. J. Med. Sci. 2005, 35, 289295.
[19] Chwatko, G., Bald, E., J. Chromatogr. A 2002, 949, 141
151.
[20] Accinni, R., Campolo, J., Bartesaghi, S., De Leo, G., Lucarelli, C., Cursano, C. F., Parodi, O., J. Chromatogr. A
1998, 828, 397400.
[21] Nekrassova, O., Lawrence, N. S., Compton, R. G., Talanta
2003, 60, 10851095.
[22] Yoshida, Y., Ohiwa, Y., Shimamura, M., Izumi, T., Yoshida,
S., Takahashi, K., Miyairi, S., Makimura, M., Naganuma,
A., J. Health Sci. 2003, 49, 527530.
[23] Gilfix, B. M., Blank, D. W., Rosenblatt, D. S., Clin. Chem.
1997, 43, 687688.
M., Kozich, V., Clin. Chem. 2001, 47,
[24] Krijt, J., Vackova,
18211828.
[25] Shabir, G. A., J. Chromatogr. A 2003, 987, 5766.
[26] International Conference on Harmonization (ICH), Guidance for Industry Q2b: Validation of Analytical Procedures: Methodology, US FDA Federal Register, Rockville
1997.

[3] Chou, S. T., Ko, L. E., Lim, P. S., Huang, J. L., Yang, C. S.,
Chin. J. Physiol. 2000, 43, 5964.

[27] Center for Drug Evaluation and Research, Reviewer

Guidance: Validation of Chromatographic Methods, US
FDA Federal Register, Rockville 1994.

[4] Bree, A., Verschuren, W. M. M., Kromhout, D., Kluijtmans, L. A. J., Blom, H. J., Pharmacol. Rev. 2002, 54,
599618.

[28] International Conference on Harmonization (ICH), Guidance for Industry: Bioanalytical Method Validation, US
FDA Federal Register, Rockville 2001.

[5] Kazemi, M. B. S., Eshraghian, K., Omrani, G. R.,

Lankarani, K. B., Hosseini, E., Iran, S., Angiology 2006,
57, 914.

[29] Garcia, A. J., Apitz-Castro, R., J. Chromatogr. B 2002,

779, 359363.

[6] Jacobsen, D. W., Clin. Chem. 1998, 44, 18331843.

[7] Chango, A., Perrin, M. O., Tronel, H., Nicolas, J. P., Nutr.
Clin. Metab. 1997, 11, 201211.

[8] Refsum, H., Ueland, P. M., Nygard,

O., Vollset, S. E.,
Annu. Rev. Med. 1998, 49, 3162.
[9] Refsum, H., Smith, A. D., Ueland, P. M., Nexo, E., Clarke,
R., Mcpartlin, J., Johnston, C., Engbaek, F., Schneede, J.,
McPartlin, C., Scott, J. M., Clin. Chem. 2004, 50, 332.

[10] Ozkan,
Y., Ozkan,
E., Simsek, B., Int. J. Cardiol. 2002, 82,
269277.
[11] Lawrence de Koning, A. B., Werstuck, G. H., Zhou, J.,
Austin, R. C., Clin. Biochem. 2003, 36, 431441.


C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

[30] Gautier, F. C., Berneron, C., Douce, P. J., Biomed. Chromatogr. 1999, 13, 239243.
[31] Toyooka, T., Imai, K., Analyst 1984, 109, 10031007.
[32] Daskalakis, I., Lucock, M. D., Anderson, A., Wild, J.,
Schorah, C. J., Levene, M. I., Biomed. Chromatogr. 1996,
10, 205212.
[33] Rizzo, V., Montalbetti, L., Valli, M., Bosoni, T., Scoglio, E.,
Moratti, R., J. Chromatogr. B 1998, 706, 209215.
[34] Fermo, I., Arcelloni, C., Mazzola, G., DAngelo, A., Paroni,
R., J. Chromatogr. B 1998, 719, 3136.

[35] Frick, B., Schrocksnadel,

K., Neurauter, G., Wirleitner, B.,
Artner-Dworzak, E., Fuchs, D., Clin. Chim. Acta 2003, 331,
1923.

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